Compositions and methods for diagnosing and treating an inflammation

ABSTRACT

A method of reducing an inflammatory response in a subject is provided. The method comprising providing to a subject in need thereof a therapeutically effective amount of an agent capable of reducing activity and/or expression of a scavenger receptor or of an effector thereof, thereby reducing the inflammatory response in the subject.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.13/721,309 filed on Dec. 20, 2012, which is a division of U.S. patentapplication Ser. No. 13/205,665 filed on Aug. 9, 2011, now U.S. Pat. No.8,409,569, which is a continuation of U.S. patent application Ser. No.11/222,745 filed on Sep. 12, 2005, now U.S. Pat. No. 8,017,113, which isa Continuation-In-Part (CIP) of PCT Patent Application No.PCT/IL2004/000241 filed on Mar. 14, 2004, which claims the benefit ofpriority from U.S. Provisional Patent Application Nos. 60/453,512 and60/453,514 both filed on Mar. 12, 2003. The contents of the aboveapplications are incorporated herein by reference.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 56516SequenceListing.txt, created on Jun. 9,2013, comprising 13,687 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to antibodies, compositions and methodsfor diagnosing and treating inflammation. More particularly, the presentinvention relates to the use of scavenger receptor inhibitors intreatment of an inflammatory response and to methods of diagnosing aninflammatory response via detection of autoantibodies directed atscavenger receptors.

Inflammation is a physiological condition characterized in the acuteform by the classical signs of pain, heat, redness, swelling and loss offunction. Inflammation often accompanies diseases such as MultipleSclerosis (MS), osteoarthritis, Inflammatory Bowl Disease (IBD)including Crohn's disease and ulcerative colitis, Rheumatoid Arthritis(RA), SLE, type I diabetes (IDDM), atherosclerosis, encephalomyelitis,Alzheimer's disease, stroke, traumatic brain injury, Parkinson'sdisease, septic shock and others. In most cases, there is no effectivecure for inflammation associated with such disease and existingtreatments are palliative and largely fail to control the underlyingcauses of tissue degradation.

Scavenger receptors (SRs) are cell surface proteins, which are typicallyfound on macrophages and bind various types of chemically modifiedlipoproteins (1-3), such as low-density lipoprotein (LDL). This familyof trans-membrane receptors which are highly varied in structure areinvolved in receptor-mediated endocytosis, phagocytosis of apoptoticcells and bacteria, as well as in cell adhesion [Peiser L. et al., Curr.Opin. Immun. 14(1):123-128, 2002]. Since the massive receptor-mediateduptake of cholesterol from modified LDL can convert cultured macrophagesinto cholesteryl ester-filled foam cells, similar to those found inatherosclerotic plaques, it has been postulated that these receptorsalso function in deposition of LDL cholesterol of macrophages in arterywalls during the initial stages of atherosclerotic plaque formation [1].

Scavenger receptors (SRs) are termed as such since they mediate thebinding of remarkably wide variety of polyanionic ligands [e.g.,modified proteins, sulfated polysaccharides and certain polynucleotides[1, 3, 4]. This property led to the hypothesis that these receptors forma part of an in innate immune response by serving as pattern recognitionreceptors that bind a wide variety of pathogen components [2-5].

SR-B1 (also referred to as SR-BI or CLA-I) is a macrophage scavengermolecule and a receptor for high-density lipoprotein (HDL) [2, 3, 6, 7]that mediates cholesterol uptake from cells [Rigotti A. et al., Curr.Opin. Lipidol., 8:181-8, 1997; Rigotti A. et al., Proc. Natl. Acad.Sci., 94:12610-5, 1997]. SR-B1 can also serve as a receptor for non-HDLlipoproteins and appears to play an important role in reversecholesterol transport. In vivo experiments showed that this receptor isimportant for HDL metabolism in mice, and for the metabolism of LDL andHDL cholesterol in humans [Stang H. et al., J. Biol. Chem. 274:32692-8.,1999; Swarnakar S. et al., J. Biol. Chem. 274:29733-9., 1999]. Studiesinvolving the manipulation of SR-B1 gene expression in mice, indicatethat its expression protects against atherosclerosis [Kozarsky K. F.,and Krieger M., Curr. Opin. Lipidol. 10:491-7., 1999; Ueda Y. et al., J.Biol. Chem. 275:20368-73., 2000; Acton S. L. et al., Mol. Med. Today5:518-24., 1999]. It was also suggested that HDL and particularly itsprotein fraction Apo-A1 affect the in vitro production ofpro-inflammatory mediators by macrophages (8). Among mediators derivedfrom macrophages that propagate inflammation are interleukin 12 (IL-12),TNF-α and possibly IL-6 whereas, TGF-β and IL-10 have predominantlyanti-inflammatory effects [Kiefer R. et al., Prog. Neurobiol.64(2):109-27, 2001].

PCT Publication No. WO 2004/041179 teaches targeting of scavengerreceptor SR-B1 (Cla-I) for the treatment of infection, sepsis andinflammation. This prior art teaches primarily targeting SR-B1 usingamphipathic peptides which compete the amphipathic helices inapoliprotein ligands of SR-B1. PCT Publication No. WO 2004/041179 doesnot provide experimental results for treating autoimmune diseases suchas IBD and multiple sclerosis by down-regulating activity or expressionof SR-B1, nor does it teach the use of oligonucleotide technology (e.g.,antisense, siRNA) and DNA vaccination for targeting SR-B1 and treatinginflammatory diseases.

There is thus, a widely recognized need for and it would be highlyadvantageous to have novel agents and methods using same for targetingSR-B1 and treating inflammatory diseases.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of reducing an inflammatory response in a subject, the methodcomprising providing to a subject in need thereof a therapeuticallyeffective amount of an agent capable of reducing activity and/orexpression of a scavenger receptor or of an effector thereof, therebyreducing the inflammatory response in the subject.

According to further features in preferred embodiments of the inventiondescribed below, the agent is selected from the group consisting of: (i)an oligonucleotide directed to an endogenous nucleic acid sequenceexpressing the scavenger receptor or the effector thereof; (ii) achemical inhibitor directed to the scavenger receptor or the effectorthereof; (iii) a neutralizing antibody directed at the scavengerreceptor or the effector thereof; (iv) a non-functional derivative ofthe scavenger receptor or the effector thereof; and (v) a DNA vaccine toscavenger receptor or the effector thereof.

According to still further features in the described preferredembodiments the scavenger receptor is a class A scavenger receptor or aclass B scavenger receptor.

According to still further features in the described preferredembodiments the class B scavenger receptor is SR-BI.

According to another aspect of the present invention there is provideduse of an agent capable of reducing activity and/or expression ofscavenger receptor or of an effector thereof for the manufacture of amedicament for the treatment of inflammatory diseases.

According to yet another aspect of the present invention there isprovided an article of manufacture comprising packaging material and apharmaceutical composition identified for treating inflammatory diseasesbeing contained within the packaging material, the pharmaceuticalcomposition including, as an active ingredient, an agent capable ofreducing activity and/or expression of scavenger receptor or of aneffector thereof and a pharmaceutically acceptable carrier.

According to still another aspect of the present invention there isprovided a method of diagnosing predisposition to, or presence of, aninflammatory disease in a subject, the method comprising detecting antiscavenger receptor antibodies in a biological sample obtained from thesubject, wherein a level above a predetermined normal threshold of theanti scavenger receptor antibodies in the biological sample isindicative of the inflammatory disease in the subject.

According to still further features in the described preferredembodiments detecting the anti scavenger receptor antibodies in thebiological sample is effected by ELISA, RIA and/or dot blot.

According to an additional aspect of the present invention there isprovided a humanized antibody having an antigen recognition domaincapable of specifically binding a scavenger receptor.

According to yet an additional aspect of the present invention there isprovided an isolated polypeptide comprising an antigen recognitiondomain capable of specifically binding a human scavenger receptor andneutralize an activity thereof.

According to still further features in the described preferredembodiments the polypeptide is an antibody or an antibody fragment.

According to still further features in the described preferredembodiments the antibody or antibody fragment is humanized.

According to still further features in the described preferredembodiments the antibody or the antibody fragment is selected from thegroup consisting of a Fab fragment, an Fv fragment, a single chainantibody and a single domain antibody.

According to still further features in the described preferredembodiments the polypeptide is a CDR-containing recombinant polypeptide.

According to still further features in the described preferredembodiments an amino acid sequence of the CDR is selected from the groupconsisting of SEQ ID NO: 11, 15, 19, 25, 29 and 33.

According to still an additional aspect of the present invention thereis provided an isolated polynucleotide comprising a nucleic acidsequence encoding the recombinant polypeptide.

According to still further features in the described preferredembodiments the antigen recognition domain is capable of specificallyrecognizing a surface exposed epitope of Scavenger Receptor.

According to a further aspect of the present invention there is provideda pharmaceutical composition comprising as an active ingredient thepolypeptide comprising an antigen recognition domain capable ofspecifically binding a human scavenger receptor and neutralize anactivity thereof.

According to a further aspect of the present invention there is provideda method of reducing an inflammatory response in a subject, the methodcomprising providing to a subject in need thereof a therapeuticallyeffective amount of the polypeptide comprising an antigen recognitiondomain capable of specifically binding a human scavenger receptor andneutralize an activity thereof, thereby reducing the inflammatoryresponse in the subject.

According to yet a further aspect of the present invention there isprovided a method of treating IBD in a subject, the method comprisingproviding to a subject in need thereof a therapeutically effectiveamount of an agent capable of reducing activity and/or expression of ascavenger receptor or of an effector thereof, thereby treating the IBDin the subject.

According to still a further aspect of the present invention there isprovided a method of treating multiple sclerosis in a subject, themethod comprising providing to a subject in need thereof atherapeutically effective amount of an agent capable of reducingactivity and/or expression of a scavenger receptor or of an effectorthereof, thereby treating multiple sclerosis in the subject.

According to still a further aspect of the present invention there isprovided a method of treating IBD in a subject, the method comprisingproviding to a subject in need thereof a therapeutically effectiveamount of the polypeptide comprising an antigen recognition domaincapable of specifically binding a human scavenger receptor andneutralize an activity thereof, thereby treating the IBD in the subject.

According to still a further aspect of the present invention there isprovided a method of treating multiple sclerosis in a subject, themethod comprising providing to a subject in need thereof atherapeutically effective amount of the polypeptide comprising anantigen recognition domain capable of specifically binding a humanscavenger receptor and neutralize an activity thereof, thereby treatingthe multiple sclerosis in the subject.

According to still a further aspect of the present invention there isprovided use of the polypeptide comprising an antigen recognition domaincapable of specifically binding a human scavenger receptor andneutralize an activity thereof for the manufacture of a medicamentidentified for treating IBD.

According to still a further aspect of the present invention there isprovided use of the polypeptide comprising an antigen recognition domaincapable of specifically binding a human scavenger receptor andneutralize an activity thereof for the manufacture of a medicamentidentified for treating multiple sclerosis.

According to still a further aspect of the present invention there isprovided a CDR-containing polypeptide having a CDR sequence selectedfrom the group consisting of SEQ ID NO: 11, 15, 19, 25, 29 and 33.

According to still a further aspect of the present invention there isprovided an isolated polynucleotide encoding the CDR-containingpolypeptide having a CDR sequence selected from the group consisting ofSEQ ID NO: 11, 15, 19, 25, 29 and 33.

According to still further features in the described preferredembodiments the isolated polynucleotide comprises a nucleic acidsequence selected from the group consisting of SEQ ID NO: 12, 16, 20,26, 30 and 32.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing novel compositions andmethods containing same for diagnosing and treating an inflammatoryresponse.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1 a-d are graphs depicting the therapeutic effect of recombinantSR-B1 injection on EAE-induced Lewis rats. FIG. 1 a is a plot graphdepicting the daily development of clinical manifestations of EAE inLewis rats previously subjected to SR-B1 vaccination. The plots comparerats (6 Lewis rats in each group) subjected to active induction of EAE,two months following last administration of plasmid DNA encoding SR-B1(opened triangles), plasmid DNA encoding β-actin (opened squares), orPBS (closed squares). Data was obtained by an observer blind to theexperimental protocol using discrete scoring (0-4) of EAE clinicalmanifestations as described in Example 1 of the Examples section.Results are presented as mean maximal score ±SE. FIG. 1 b is a bar graphdepicting antibody titer in Lewis rats sera in response toadministration of recombinant SR-B1. Lewis rats were subjected torepeated administration of plasmid DNA encoding SR-B1, plasmid DNAencoding β-actin, or PBS (none). Two months after the last immunizationall rats were subjected to active induction of EAE and sera was takenafter 13 days and determined for the development of antibody titer torecombinant SR-B1. Data was obtained by ELISA, in which rat anti-sera,detected the recombinant SR-B1 (OR soluble β-ACTIN) coated onto 96 wellELISA plates. Antibodies were labeled with Goat anti-rat IgG alkalinephosphatase conjugated antibody and in the presence of p-NitrophenylPhosphate (p-NPP) liquid substrate produces absorbance at 405 nm. Datawas collected using ELISA reader. Results are presented as log ₂-Abtiter±SE. FIG. 1 c is a plot graph depicting specific binding of antiSR-B1 antibodies to the native form of SR-B1 on activated peritonealmacrophages. The graphs represent flow cytometry analysis of ratactivated peritoneal macrophages cells (10⁶) incubated for 30 minuteswith IgG (0.5 μg/ml) labeled with anti-rat IgG-FITC, purified from ratsthat were subjected to plasmid DNA encoding SR-B1 (Anti SR-BI) or toplasmid DNA encoding β-actin (control IgG) administration. Data wascollected using a FACScalibur. Results are presented as relative cellnumber. FIG. 1 d is a plot graph depicting the daily development ofclinical manifestations of EAE in Lewis rats subjected to IgGadministration. The plots compare rats (6 Lewis rats in each group)subjected to active induction of EAE and then repeated (days 5, 7, 9)administration of 100 μg/rat of purified IgG from EAE rats treated withplasmid DNA encoding SR-B1 (opened triangles), plasmid DNA encodingβ-actin (opened squares), or PBS (closed squares). Data was obtained byan observer blind to the experimental protocol using discrete scoring(0-4) of EAE clinical manifestations as described in Example 1 of theExamples section. Results are presented as mean maximal score ±SE;

FIGS. 2 a-c are graphs depicting the effect of anti SR-B1 antibodies oncytokine expression by murine peritoneal macrophages. The plots comparelevels of IL-12 (FIG. 2 a), TNF-α (FIG. 2 b) and IL-10 (FIG. 2 c)produced by LPS activated murine peritoneal macrophages supplementedwith (0, 10, 50 or 100 μg/ml) purified anti SR-B1 polyclonalautoantibodies (closed squares), or control IgG (open squares) fromnormal rat serum after 48 h incubation. Data was obtained by ELISA usingcommercial kits. Data was collected using ELISA reader. Results arepresented in pg/ml as mean triplicates ±SE;

FIG. 3 is a graph depicting the daily development of clinicalmanifestations of EAE in C57BL/6 mice treated with SR-B1 DNA vaccine.The plots compare treatment of mice (6 in each group) with SR-B1encoding DNA vaccine (closed squares), empty plasmid (closed circles),or PBS (opened squares) 12 days after the onset of disease. Data wasobtained by an observer blind to the experimental protocol usingdiscrete scoring (0-4) of EAE clinical manifestations as described inExample 1 of the Examples section. Results are presented as mean maximalscore ±SE;

FIG. 4 is a graph depicting the daily development of clinicalmanifestations of EAE in Lewis rats treated with anti SR-B1 antibodies.The plots compare treatment of rats (6 Lewis rats in each group) with100 μg/ml of monoclonal antibody 5D8 (closed triangle), control murineIgG1 (closed circles), anti SR-B1 polyclonal antibody (closed squares),control rat IgG2a purified from EAE rats that have been subjected to anempty plasmid DNA vaccination (open circles) or PBS treated control(open squares). Data was obtained by an observer blind to theexperimental protocol using discrete scoring (0-4) of EAE clinicalmanifestations as described in Example 1 of the Examples section.Results are presented as mean maximal score ±SE;

FIGS. 5 a-d are photomicrographs depicting colon histology sections ofcolitis induced rats. FIG. 5 a is a representative histology section ofcolon of an untreated rat (normal colon). FIG. 5 b is a representativehistology section of a rat colon, 15 days post induction of colitis,subjected to repeated administration of PBS (days 6, 8 and 10 postinduction of the disease—control sick rat colon). FIG. 5 c is arepresentative histology section of a rat colon, 15 days post inductionof colitis, subjected to repeated administration of control IgG (days 6,8 and 10 post induction of the disease—control IgG treated colon). FIG.5 d is a representative histology section of a rat colon, 15 days postinduction of colitis, subjected to repeated administration of anti SR-B1polyclonal antibodies (days 6, 8 and 10 post induction of thedisease—anti SR-B1 treated colon). Photos were obtained using a digitalcamera (Nikon digital camera DXM1200F) and a light microscopy (NikonTE2000-S);

FIG. 6 is a photograph depicting cross-reactivity of monoclonal antiSR-B1 antibody, E12, with human and mouse orthologs. Recombinantproteins were resolved on SDS-PAGE and transferred to nitrocellulosemembrane. The membrane was subjected to E12;

FIG. 7 is a graph depicting dose-dependent induction of IL-10 secretionfrom cultured peritoneal macrophages treated with E12;

FIG. 8 is a bar graph depicting dose-dependent suppression of NO levelsin cultured peritoneal macrophages treated with E12;

FIG. 9 is a graph depicting the effect of E12 (closed squares), controlisotype matching antibody (circles) or no treatment on ongoing EAE inmice induced with such, as determined by reduction in EAE score;

FIGS. 10 a-c are bar graphs depicting the effect of E12 (pink) orcontrol antibodies (grey) on cytokine secretion from spleen cells of 19day EAE-induced mice. FIG. 10 a—IL-4. FIG. 10 b—IL-12. FIG. 10 c—IL-10;

FIGS. 11 a-f are photographs showing IL-10 immunostaining of Lumbarspinal cord sections from EAE induced mice (19 days of disease onset)subjected to no treatment (FIG. 11 a), or treated with E12 (FIG. 11 b),or isotype matching control antibody (FIG. 11 c). FIGS. 11 a-c showstaining with biotinylated E12 for presence of scavenger receptorexpressing cells. FIGS. 11 d-f shows staining with anti IL-10 antibody.Anti-SR-BI therapy reduces the histological score of EAE;

FIGS. 12 a-e are photographs showing representative histological colonsections obtained at day 12 of disease onset from naïve rats (FIG. 12a), positive control rats suffering from TNBS induced IBD (FIG. 12 b),rats suffering from TNBS induced IBD that were subjected to repeatedadministration of isotype matched control IgG (FIG. 12 c) in comparisonto those treated with mAb E12 (FIGS. 12 d-e); and

FIGS. 13 a-i show representative immuno-histological sections obtainedat day 12 of disease onset from control rats suffering from TNBS inducedIBD (FIGS. 13 a-c), rats suffering from TNBS induced IBD that weresubjected to repeated administration of isotype matched control IgG(FIGS. 13 d-f) in comparison to diseased rats treated with mAb E12(FIGS. 13 g-i). FIGS. 13 a, d and g are stained with mAb ED1(macrophages bio-marker); FIGS. 13 b, e and h are stained with anti CD3(T cell bio-marker) and FIGS. 13 c, f and I are stained with an antiIL-10 mAb.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of compositions and methods which can be usedfor the diagnosis and treatment of inflammation. Specifically, thepresent invention relates to the use of scavenger receptor inhibitors intreating inflammatory response and to methods of diagnosing inflammatoryresponse via detection of autoantibodies to scavenger receptors insubjects.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Diseases and disorders which have significant inflammatory componentsare ubiquitous. Skin disorders, bowel disorders, certain degenerativeneurological disorders, arthritis, autoimmune diseases and otherillnesses afflict many patients. The factors underlying these disordersare varied and include infectious agents, autoimmune factors, dietary orenvironmental factors and genetic factors. In the majority of cases, thecausative elements have not been defined and many of the keypathophysiological components have not been elucidated. Accordingly,treatment options for the majority of these diseases is suboptimal.

The present inventor has previously shown that the immune system canselectively generate autoimmunity to chemokines and otherproinflammatory mediators when such a response is beneficial for thehost [9, 10, 11, 12, 14, 15]. For example, patients suffering fromrheumatoid arthritis (RA) but not osteoarthritis (OA) have significantlevels of autoantibodies directed to TNF-α, and therapies thatneutralize the function of TNF-α suppress RA but not OA. Studiesconducted by the present inventor have shown that selectiveamplification of these beneficial antibodies by targeted DNA vaccinesprovided protective immunity in experimental models (9, 10, 11, 12, 14,15).

While reducing the present invention to practice, the present inventoruncovered that subjects suffering from inflammatory disease exhibitelevated levels of autoantibodies to scavenger receptor (SR) and showedthat inhibiting SR function can prevent such diseases by altering thecytokine profile produced by macrophages from pro-inflammatory cytokinesto anti-inflammatory cytokines.

As is illustrated hereinbelow and in the Examples section which follows,the present inventors were able to show that anti SR-BI (CLA-I) therapy(e.g., DNA vaccination, and antibody therapy) can be used to suppressongoing inflammatory diseases such as experimental autoimmuneencephalomyelitis (EAE) and Intestinal Bowels Disease (IBD, see Examples1-5). The present inventors also developed, through laboriousexperimentations and screening a novel therapeutic anti-SR-B1 monoclonalantibody, E12, which is capable of altering the cytokine profile andinflammatory activities of macrophages. This antibody which is directedagainst a surface exposed epitope of the scavenger receptor (FIG. 6) iscross-reactive to human CLA-I (human SR-B1) and also affects thecytokine profile and in vitro activities of human macrophages (a cellline) and as such can be used as a valuable therapeutic tool (seeExample 6). This antibody was also shown effective in suppressingongoing EAE and TNBS induced IBD (see Example 7). Immunohistologicalanalysis clearly showed that in both diseases anti SR-BI therapy alteredthe cytokine production of invading leukocytes, at the autoimmune site,into high IL-10 producing cells. This may explain, significanttherapeutic effect of this antibody in these diseases.Immunohistological analysis of CNS sections using anti SR-BI mAb alsoshowed that SR-BI positive leukocytes enter the site of inflammation (sofar detected only for EAE). Thus, it is suggested that anti SR-BIantibodies affect the cytokine profile and inflammatory functions ofinflammatory leukocytes (mostly monocytes) entering the autoimmune site,and thereby the function and polarization of autoimmune T cells there.

These findings suggest that scavenger receptors can be used as targetsfor diagnostics and treatment of inflammatory diseases, especially IBDand multiple sclerosis.

Thus, according to one aspect of the present invention there is provideda method of reducing an inflammatory response in a subject.

As used herein the term “reducing” refers to preventing, curing,reversing, attenuating, alleviating, minimizing, suppressing or haltingthe deleterious effects of an inflammatory response.

As used herein the phrase “inflammatory response” refers to an immuneresponse which results in inflammation, typically occurring as a resultof injurious stimuli including infection, burns, trauma, neoplasia,autoimmune signals and exposure to chemicals, heat or cold or any otherharmful stimulus. An inflammatory response according to the presentinvention refers to an acute phase response and a chronic inflammation.

As used herein the term “subject” refers to subject who may benefit fromthe present invention such as a mammal (e.g., canine, feline, ovine,porcine, equine, bovine, human), preferably a human subject.

The method of this aspect of the present invention is effected byproviding to a subject in need thereof a therapeutically effectiveamount of an agent capable of reducing activity and/or expression of ascavenger receptor or of an effector thereof, thereby reducing theinflammatory response in the subject.

As is described in detail hereinbelow, such an agent can directly reduceactivity and/or expression of the scavenger receptor, or alternativelyactivate endogenous components which in turn reduce activity and/orexpression of the scavenger receptor (indirect).

As used herein a “scavenger receptor” refers to a gene product (i.e.,RNA or protein) of a scavenger receptor, which is known in the Art.Examples of scavenger receptors include but are not limited to class Ascavenger receptors, class B scavenger receptors and class F scavengerreceptors. The scavenger receptor is preferably one which is expressedand displayed by macrophages. Preferably, the scavenger receptor of thepresent invention is SR-BI, a member of the CD36 family, GenBankAccession No. NP_(—)005496, also known as CLA-I or SR-B1.

Scavenger receptor activity refers to cell adhesion activity,transporter activity, apoptotic activity, lipid metabolism activity,signal transduction activity and/or preferably cytokine secretionactivity.

An effector of a scavenger receptor refers to an endogenous moleculewhich up-regulates or activates scavenger receptor activity. Forexample, an effector can be a modified lipid (e.g., oxidized lipid,glycated lipid, alkylated lipid, nitrated lipid, acetylated lipid),which binds to the scavenger receptor and activates signaling therefrom.

A number of agents can be used in accordance with this aspect of thepresent invention to reduce the activity or expression of a scavengerreceptor or an effector thereof. Depending on the type of moleculeutilized, an agent can either be directly administered to the subject orexpressed in cells thereof as is further described hereinbelow.

Thus, for example the agent can be a complementarity-determining region(CDR) containing polypeptide (e.g., a neutralizing antibody) whichinhibits the activity of a scavenger receptor [such as by binding to theextracellular collagenous domain of SR which plays a role in ligandbinding. See Acton (1993) J. Biol. Chem. 268(5):3530-7] or an effectorthereof. Also provided are polynucleotides encoding such CDR containingpolypeptides. A scavenger receptor neutralizing antibody is described inExample 6 of the Examples section which follows. Other SR-neutralizingantibodies are known in the art, see for example Frolov (2000) J. Biol.Chem. 275(17): 12769-12780. According to presently known preferredembodiments the neutralizing antibody comprises an antigen recognitiondomain comprising at least one CDR selected from the group consisting ofSEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36(e.g., E12, see Example 6).

The term “antibody” refers to whole antibody molecules as well asfunctional fragments thereof, such as Fab, F(ab′)₂, and Fv that arecapable of binding with antigenic portions of the target polypeptide.These functional antibody fragments constitute preferred embodiments ofthe present invention, and are defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule that can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule as described in, for example,U.S. Pat. No. 4,946,778.

Purification of serum immunoglobulin antibodies (polyclonal antisera) orreactive portions thereof can be accomplished by a variety of methodsknown to those of skill including, precipitation by ammonium sulfate orsodium sulfate followed by dialysis against saline, ion exchangechromatography, affinity or immunoaffinity chromatography as well as gelfiltration, zone electrophoresis, etc. (see Goding in, MonoclonalAntibodies: Principles and Practice, 2nd ed., pp. 104-126, 1986,Orlando, Fla., Academic Press). Under normal physiological conditionsantibodies are found in plasma and other body fluids and in the membraneof certain cells and are produced by lymphocytes of the type denoted Bcells or their functional equivalent. Antibodies of the IgG class aremade up of four polypeptide chains linked together by disulfide bonds.The four chains of intact IgG molecules are two identical heavy chainsreferred to as H-chains and two identical light chains referred to asL-chains. Additional classes include IgD, IgE, IgA, IgM and relatedproteins.

Methods of generating and isolating monoclonal antibodies are well knownin the art, as summarized for example in reviews such as Tramontano andSchloeder, Methods in Enzymology 178, 551-568, 1989. A recombinantscavenger receptor polypeptide may be used to generate antibodies invitro (see Example 6 of the Examples section which follows). In general,a suitable host animal is immunized with the recombinant polypeptide.Advantageously, the animal host used is a mouse of an inbred strain.Animals are typically immunized with a mixture comprising a solution ofthe recombinant polypeptide in a physiologically acceptable vehicle, andany suitable adjuvant, which achieves an enhanced immune response to theimmunogen. By way of example, the primary immunization conveniently maybe accomplished with a mixture of a solution of the recombinantpolypeptide and Freund's complete adjuvant, said mixture being preparedin the form of a water in oil emulsion. Typically the immunization willbe administered to the animals intramuscularly, intradermally,subcutaneously, intraperitoneally, into the footpads, or by anyappropriate route of administration. The immunization schedule of theimmunogen may be adapted as required, but customarily involves severalsubsequent or secondary immunizations using a milder adjuvant such asFreund's incomplete adjuvant. Antibody titers and specificity of bindingto the polypeptide can be determined during the immunization schedule byany convenient method including by way of example radioimmunoassay, orenzyme linked immunosorbant assay, which is known as the ELISA assay.When suitable antibody titers are achieved, antibody-producinglymphocytes from the immunized animals are obtained, and these arecultured, selected and cloned, as is known in the art. Typically,lymphocytes may be obtained in large numbers from the spleens ofimmunized animals, but they may also be retrieved from the circulation,the lymph nodes or other lymphoid organs. Lymphocytes are then fusedwith any suitable myeloma cell line, to yield hybridomas, as is wellknown in the art. Alternatively, lymphocytes may also be stimulated togrow in culture, and may be immortalized by methods known in the artincluding the exposure of these lymphocytes to a virus, a chemical or anucleic acid such as an oncogene, according to established protocols.After fusion, the hybridomas are cultured under suitable cultureconditions, for example in multi-well plates, and the culturesupernatants are screened to identify cultures containing antibodiesthat recognize the hapten of choice. Hybridomas that secrete antibodiesthat recognize the recombinant polypeptide are cloned by limitingdilution and expanded, under appropriate culture conditions. Monoclonalantibodies are purified and characterized in terms of immunoglobulintype and binding affinity.

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment.

Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly. These methods are described, for example,by Goldenberg, in U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety (see also Porter, R. R., Biochem. J., 73: 119-126, 1959).Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of V_(H) and V_(L) chains. Thisassociation may be noncovalent, as described in Inbar et al. (Proc.Nat'l Acad. Sci. USA 69:2659-62, 1972). Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise V_(H) and V_(L) chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the V_(H) and V_(L)domains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426,1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al.,U.S. Pat. No. 4,946,778, all of which are hereby incorporated byreference in its entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick and FryMethods, 2: 106-10, 1991).

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues form a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues, which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin [Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art(see also Example 6 of the Examples section). Generally, a humanizedantibody has one or more amino acid residues introduced into it from asource, which is non-human. These non-human amino acid residues areoften referred to as import residues, which are typically taken from animport variable domain. Humanization can be essentially performedfollowing the method of Winter and co-workers [Jones et al., Nature,321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodentCDRs or CDR sequences for the corresponding sequences of a humanantibody. Accordingly, such humanized antibodies are chimeric antibodies(U.S. Pat. No. 4,816,567), wherein substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites in rodentantibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human monoclonal antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

An agent for reducing the activity of a scavenger receptor or aneffector thereof can also be a non-functional derivative thereof (i.e.,dominant negative). For example, artificial dominant negative moleculesof scavenger receptors have been previously described by Acton (1993Supra. Such truncation mutants lack the positively charged collagenousextracellular domain and while retain trimerization, post-translationalprocessing, intracellular transport, surface expression, and stability,are unable to bind ligand and have a dominant negative effects overwild-type receptors [see also Dejager et al. J Clin Invest. 1993 August;92(2):894-902].

It will be appreciated that when available, naturally occurringnon-functional derivatives of the pathway can be used. Thus, forexample, the present invention can use the natural inhibitor of SR-Aisoform which modifies ligand uptake [see Gough J Lipid Res. 1998 March;39(3):531-43].

Polypeptides of these non-functional derivatives can be synthesizedusing solid phase peptide synthesis procedures which are well known inthe art and further described by John Morrow Stewart and Janis DillahaYoung, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company,1984). Synthetic peptides can be purified by preparative highperformance liquid chromatography [Creighton T. (1983) Proteins,structures and molecular principles. WH Freeman and Co. N.Y.] and thecomposition of which can be confirmed via amino acid sequencing.

In cases where large amounts of the proteins are desired, they can begenerated using recombinant techniques such as described by Bitter etal., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990)Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514,Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J.3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al.(1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988,Methods for Plant Molecular Biology, Academic Press, NY, Section VIII,pp 421-463.

Alternatively, these proteins can encoded expressed within the targetcell from an exogenous polynucleotides ligated into a nucleic acidexpression construct.

It will be appreciated that the nucleic acid construct can beadministered to the individual employing any suitable mode ofadministration, described hereinbelow (i.e., in vivo gene therapy).Alternatively, the nucleic acid construct is introduced into a suitablecell via an appropriate gene delivery vehicle/method (transfection,transduction, homologous recombination, etc.) and an expression systemas needed and then the modified cells are expanded in culture andreturned to the individual (i.e., ex vivo gene therapy).

To enable cellular expression of the polynucleotides of the presentinvention, the nucleic acid construct of the present invention furtherincludes at least one cis acting regulatory element. As used herein, thephrase “cis acting regulatory element” refers to a polynucleotidesequence, preferably a promoter, which binds a trans acting regulatorand regulates the transcription of a coding sequence located downstreamthereto.

Any suitable promoter sequence can be used by the nucleic acid constructof the present invention.

Preferably, the promoter utilized by the nucleic acid construct of thepresent invention is active in the specific cell population transformed.Examples of cell type-specific and/or tissue-specific promoters includepromoters such as albumin that is liver specific [Pinkert et al., (1987)Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al.,(1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cellreceptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins;[Banerji et al. (1983) Cell 33729-740], neuron-specific promoters suchas the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad.Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al.(1985) Science 230:912-916] or mammary gland-specific promoters such asthe milk whey promoter (U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). The nucleic acid construct of the presentinvention can further include an enhancer, which can be adjacent ordistant to the promoter sequence and can function in up regulating thetranscription therefrom.

The nucleic acid construct of the present invention preferably furtherincludes an appropriate selectable marker and/or an origin ofreplication. Preferably, the nucleic acid construct utilized is ashuttle vector, which can propagate both in E. coli (wherein theconstruct comprises an appropriate selectable marker and origin ofreplication) and be compatible for propagation in cells, or integrationin a gene and a tissue of choice. The construct according to the presentinvention can be, for example, a plasmid, a bacmid, a phagemid, acosmid, a phage, a virus or an artificial chromosome.

Examples of suitable constructs include, but are not limited to, pcDNA3,pcDNA3.1 (+/−), pGL3, PzeoSV2 (+/−), pDisplay, pEF/myc/cyto,pCMV/myc/cyto each of which is commercially available from InvitrogenCo. (see Invitrogen's website). Examples of retroviral vector andpackaging systems are those sold by Clontech, San Diego, Calif.,including Retro-X vectors pLNCX and pLXSN, which permit cloning intomultiple cloning sites and the trasgene is transcribed from CMVpromoter. Vectors derived from Mo-MuLV are also included such as pBabe,where the transgene will be transcribed from the 5′LTR promoter.

Currently preferred in vivo nucleic acid transfer techniques includetransfection with viral or non-viral constructs, such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) andlipid-based systems. Useful lipids for lipid-mediated transfer of thegene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al.,Cancer Investigation, 14(1): 54-65 (1996)]. The most preferredconstructs for use in gene therapy are viruses, most preferablyadenoviruses, AAV, lentiviruses, or retroviruses. A viral construct suchas a retroviral construct includes at least one transcriptionalpromoter/enhancer or locus-defining element(s), or other elements thatcontrol gene expression by other means such as alternate splicing,nuclear RNA export, or post-translational modification of messenger.Such vector constructs also include a packaging signal, long terminalrepeats (LTRs) or portions thereof, and positive and negative strandprimer binding sites appropriate to the virus used, unless it is alreadypresent in the viral construct. In addition, such a construct typicallyincludes a signal sequence for secretion of the peptide from a host cellin which it is placed. Preferably, the signal sequence for this purposeis a mammalian signal sequence or the signal sequence of the polypeptidevariants of the present invention. Optionally, the construct may alsoinclude a signal that directs polyadenylation, as well as one or morerestriction sites and a translation termination sequence. By way ofexample, such constructs will typically include a 5′ LTR, a tRNA bindingsite, a packaging signal, an origin of second-strand DNA synthesis, anda 3′ LTR or a portion thereof. Other vectors can be used that arenon-viral, such as cationic lipids, polylysine, and dendrimers.

Alternatively, the agent of this aspect of the present invention can bea chemical, which is designed to specifically inhibit the activity orexpression of a scavenger receptor or an effector thereof. An example ofa scavenger receptor inhibitor is Pitavastatin [NK-104, Circulation.2004 Feb. 17; 109(6):790-6], which down-regulates expression of CD36.Further it is well established that TNF-α regulates scavenger receptorexpression through MAPK (e.g., ERK, JNK, and p38). Thus it is suggestedthat short term inhibition of this pathway down-regulates the receptorexpression, while long term treatment with TNF-α is expected to have thesame effect [Hsu J Biol Chem. 2000 Dec. 29; 275(52):41035-48]. Signaltransduction factors and inhibitors are available from a number ofchemical companies including Calbiochem (San Diego, Calif., USA) andSigma-Aldrich Corp. (St Louis, Mo., USA).

Another agent capable of reducing the expression of an SR or effectorsthereof is a small interfering RNA (siRNA) molecule. RNA interference isa two-step process. the first step, which is termed as the initiationstep, input dsRNA is digested into 21-23 nucleotide (nt) smallinterfering RNAs (siRNA), probably by the action of Dicer, a member ofthe RNase III family of dsRNA-specific ribonucleases, which processes(cleaves) dsRNA (introduced directly or via a transgene or a virus) inan ATP-dependent manner. Successive cleavage events degrade the RNA to19-21 bp duplexes (siRNA), each with 2-nucleotide 3′ overhangs[Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232(2002); and Bernstein Nature 409:363-366 (2001)].

In the effector step, the siRNA duplexes bind to a nuclease complex tofrom the RNA-induced silencing complex (RISC). An ATP-dependentunwinding of the siRNA duplex is required for activation of the RISC.The active RISC then targets the homologous transcript by base pairinginteractions and cleaves the mRNA into 12 nucleotide fragments from the3′ terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12:225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen.2:110-119 (2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although themechanism of cleavage is still to be elucidated, research indicates thateach RISC contains a single siRNA and an RNase [Hutvagner and ZamoreCurr. Opin. Genetics and Development 12:225-232 (2002)].

Because of the remarkable potency of RNAi, an amplification step withinthe RNAi pathway has been suggested. Amplification could occur bycopying of the input dsRNAs which would generate more siRNAs, or byreplication of the siRNAs formed. Alternatively or additionally,amplification could be effected by multiple turnover events of the RISC[Hammond et al. Nat. Rev. Gen. 2:110-119 (2001), Sharp Genes. Dev.15:485-90 (2001); Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12:225-232 (2002)]. For more information on RNAi see thefollowing reviews Tuschl ChemBiochem. 2:239-245 (2001); Cullen Nat.Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys. Act. 1575:15-25(2002).

Synthesis of RNAi molecules suitable for use with the present inventioncan be effected as follows. First, an SR-B1 mRNA sequence (e.g., GenBankAccession No. NP_(—)005496), for example, is scanned downstream of theAUG start codon for AA dinucleotide sequences. Occurrence of each AA andthe 3′ adjacent 19 nucleotides is recorded as potential siRNA targetsites. Preferably, siRNA target sites are selected from the open readingframe, as untranslated regions (UTRs) are richer in regulatory proteinbinding sites. UTR-binding proteins and/or translation initiationcomplexes may interfere with binding of the siRNA endonuclease complex[Tuschl ChemBiochem. 2:239-245]. It will be appreciated though, thatsiRNAs directed at untranslated regions may also be effective, asdemonstrated for GAPDH wherein siRNA directed at the 5′ UTR mediatedabout 90% decrease in cellular GAPDH mRNA and completely abolishedprotein level (see Ambion's website).

Second, potential target sites are compared to an appropriate genomicdatabase (e.g., human, mouse, rat etc.) using any sequence alignmentsoftware, such as the BLAST software available from the NCBI server ( ).Putative target sites which exhibit significant homology to other codingsequences are filtered out.

Qualifying target sequences are selected as template for siRNAsynthesis. Preferred sequences are those including low G/C content asthese have proven to be more effective in mediating gene silencing ascompared to those with G/C content higher than 55%. Several target sitesare preferably selected along the length of the target gene forevaluation.

For better evaluation of the selected siRNAs, a negative control ispreferably used in conjunction. Negative control siRNA preferablyinclude the same nucleotide composition as the siRNAs but lacksignificant homology to the genome. Thus, a scrambled nucleotidesequence of the siRNA is preferably used, provided it does not displayany significant homology to any other gene.

One example of a siRNA molecule directed at a SR (i.e., p120) isdescribed by Nishikawa Eur J Biochem. 2001 October; 268(20):5295-9.

Another agent capable of downregulating a SR or an effector thereof is aDNAzyme molecule capable of specifically cleaving an mRNA transcript orDNA sequence of interest. DNAzymes are single-stranded polynucleotideswhich are capable of cleaving both single and double stranded targetsequences (Breaker, R. R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997;943:4262) A general model (the “10-23” model) for the DNAzyme has beenproposed. “10-23” DNAzymes have a catalytic domain of 15deoxyribonucleotides, flanked by two substrate-recognition domains ofseven to nine deoxyribonucleotides each. This type of DNAzyme caneffectively cleave its substrate RNA at purine:pyrimidine junctions(Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for revof DNAzymes see Khachigian, L M [Curr Opin Mol Ther 4:119-21 (2002)].

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single and double-stranded target cleavage siteshave been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymesof similar design directed against the human Urokinase receptor wererecently observed to inhibit Urokinase receptor expression, andsuccessfully inhibit colon cancer cell metastasis in vivo (Itoh et al,20002, Abstract 409, Ann Meeting Am Soc Gen Ther). In anotherapplication, DNAzymes complementary to bcr-ab1 oncogenes were successfulin inhibiting the oncogenes expression in leukemia cells, and lesseningrelapse rates in autologous bone marrow transplant in cases of CML andALL.

Reducing expression of SR or an effector thereof can also be effected byusing an antisense polynucleotide capable of specifically hybridizingwith an mRNA transcript encoding the proteins of interest.

Design of antisense molecules which can be used to efficientlydownregulate a gene product of interest must be effected whileconsidering two aspects important to the antisense approach. The firstaspect is delivery of the oligonucleotide into the cytoplasm of theappropriate cells, while the second aspect is design of anoligonucleotide which specifically binds the designated mRNA withincells in a way which inhibits translation thereof.

The prior art teaches of a number of delivery strategies which can beused to efficiently deliver oligonucleotides into a wide variety of celltypes [see, for example, Luft J Mol Med 76: 75-6 (1998); Kronenwett etal. Blood 91: 852-62 (1998); Rajur et al. Bioconjug Chem 8: 935-40(1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71 (1997) andAoki et al. (1997) Biochem Biophys Res Commun 231: 540-5 (1997)].

In addition, algorithms for identifying those sequences with the highestpredicted binding affinity for their target mRNA based on athermodynamic cycle that accounts for the energetics of structuralalterations in both the target mRNA and the oligonucleotide are alsoavailable [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9(1999)].

Such algorithms have been successfully used to implement an antisenseapproach in cells. For example, the algorithm developed by Walton et al.enabled scientists to successfully design antisense oligonucleotides forrabbit β-globin (RBG) and mouse tumor necrosis factor-α (TNF-α)transcripts. The same research group has more recently reported that theantisense activity of rationally selected oligonucleotides against threemodel target mRNAs (human lactate dehydrogenase A and B and rat gp130)in cell culture as evaluated by a kinetic PCR technique proved effectivein almost all cases, including tests against three different targets intwo cell types with phosphodiester and phosphorothioate oligonucleotidechemistries.

In addition, several approaches for designing and predicting efficiencyof specific oligonucleotides using an in vitro system were alsopublished (Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)].

Several clinical trials have demonstrated safety, feasibility andactivity of antisense oligonucleotides. For example, antisenseoligonucleotides suitable for the treatment of cancer have beensuccessfully used [Holmund et al., Curr Opin Mol Ther 1:372-85 (1999)],while treatment of hematological malignancies via antisenseoligonucleotides targeting c-myb gene, p53 and Bcl-2 had enteredclinical trials and had been shown to be tolerated by patients [GerwitzCurr Opin Mol Ther 1:297-306 (1999)].

More recently, antisense-mediated suppression of human heparanase geneexpression has been reported to inhibit pleural dissemination of humancancer cells in a mouse model [Uno et al., Cancer Res 61:7855-60(2001)].

Thus, the current consensus is that recent developments in the field ofantisense technology which, as described above, have led to thegeneration of highly accurate antisense design algorithms and a widevariety of oligonucleotide delivery systems, enable an ordinarilyskilled artisan to design and implement antisense approaches suitablefor downregulating expression of known sequences without having toresort to undue trial and error experimentation.

SR-specific antisense molecules have been previously described byRhainds Biochemistry. 2003 Jun. 24; 42(24):7527-38; Zingg ArteriosclerThromb Vasc Biol. 2002 Mar. 1; 22(3):412-7; and Imachi Lab Invest. 2000February; 80(2):263-70.

Another agent capable of reducing the expression of scavenger receptoror an effector thereof is a ribozyme molecule capable of specificallycleaving an mRNA transcript encoding this gene product. Ribozymes arebeing increasingly used for the sequence-specific inhibition of geneexpression by the cleavage of mRNAs encoding proteins of interest [Welchet al., Curr Opin Biotechnol. 9:486-96 (1998)]. The possibility ofdesigning ribozymes to cleave any specific target RNA has rendered themvaluable tools in both basic research and therapeutic applications. Inthe therapeutics area, ribozymes have been exploited to target viralRNAs in infectious diseases, dominant oncogenes in cancers and specificsomatic mutations in genetic disorders [Welch et al., Clin Diagn Virol.10:163-71 (1998)]. Most notably, several ribozyme gene therapy protocolsfor HIV patients are already in Phase 1 trials. More recently, ribozymeshave been used for transgenic animal research, gene target validationand pathway elucidation. Several ribozymes are in various stages ofclinical trials. ANGIOZYME was the first chemically synthesized ribozymeto be studied in human clinical trials. ANGIOZYME specifically inhibitsformation of the VEGF-r (Vascular Endothelial Growth Factor receptor), akey component in the angiogenesis pathway. Ribozyme Pharmaceuticals,Inc., as well as other firms have demonstrated the importance ofanti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozymedesigned to selectively destroy Hepatitis C Virus (HCV) RNA, was foundeffective in decreasing Hepatitis C viral RNA in cell culture assays(Ribozyme Pharmaceuticals, Incorporated—WEB home page).

An additional method of reducing the expression of an SR gene oreffectors thereof in cells is via triplex forming oligonucleotides(TFOs). Recent studies have shown that TFOs can be designed which canrecognize and bind to polypurine/polypirimidine regions indouble-stranded helical DNA in a sequence-specific manner. Theserecognition rules are outlined by Maher III, L. J., et al., Science,1989; 245:725-730; Moser, H. E., et al., Science, 1987; 238:645-630;Beal, P. A., et al, Science, 1992; 251:1360-1363; Cooney, M., et al.,Science, 1988; 241:456-459; and Hogan, M. E., et al., EP Publication375408. Modification of the oligonucleotides, such as the introductionof intercalators and backbone substitutions, and optimization of bindingconditions (pH and cation concentration) have aided in overcominginherent obstacles to TFO activity such as charge repulsion andinstability, and it was recently shown that synthetic oligonucleotidescan be targeted to specific sequences (for a recent review see Seidmanand Glazer, J Clin Invest 2003; 112:487-94).

In general, the triplex-forming oligonucleotide has the sequencecorrespondence:

oligo 3′--A G G T duplex 5′--A G C T duplex 3′--T C G A

However, it has been shown that the A-AT and G-GC triplets have thegreatest triple helical stability (Reither and Jeltsch, BMC Biochem,2002, Sep. 12, Epub). The same authors have demonstrated that TFOsdesigned according to the A-AT and G-GC rule do not form non-specifictriplexes, indicating that the triplex formation is indeed sequencespecific.

Thus for any given sequence a triplex forming sequence may be devised.Triplex-forming oligonucleotides preferably are at least 15, morepreferably 25, still more preferably 30 or more nucleotides in length,up to 50 or 100 bp.

Transfection of cells (for example, via cationic liposomes) with TFOs,and formation of the triple helical structure with the target DNAinduces steric and functional changes, blocking transcription initiationand elongation, allowing the introduction of desired sequence changes inthe endogenous DNA and resulting in the specific downregulation of geneexpression. Examples of such suppression of gene expression in cellstreated with TFOs include knockout of episomal supFG1 and endogenousHPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999;27:1176-81, and Puri, et al, J Biol Chem, 2001; 276:28991-98), and thesequence- and target specific downregulation of expression of the Ets2transcription factor, important in prostate cancer etiology (Carbone, etal, Nucl Acid Res. 2003; 31:833-43), and the pro-inflammatory ICAM-1gene (Besch et al, J Biol Chem, 2002; 277:32473-79). In addition,Vuyisich and Beal have recently shown that sequence specific TFOs canbind to dsRNA, inhibiting activity of dsRNA-dependent enzymes such asRNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

Additionally, TFOs designed according to the abovementioned principlescan induce directed mutagenesis capable of effecting DNA repair, thusproviding both downregulation and upregulation of expression ofendogenous genes (Seidman and Glazer, J Clin Invest 2003; 112:487-94).Detailed description of the design, synthesis and administration ofeffective TFOs can be found in U.S. Patent Application Nos. 2003 017068and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 toEmanuele et al, and U.S. Pat. No. 5,721,138 to Lawn.

Additional description of oligonucleotide agents is further providedhereinbelow. It will be appreciated that therapeutic oligonucleotidesmay further include base and/or backbone modifications which mayincrease bioavailability therapeutic efficacy and reduce cytotoxicity.Such modifications are described in Younes (2002) Current PharmaceuticalDesign 8:1451-1466.

For example, the oligonucleotides of the present invention may compriseheterocylic nucleosides consisting of purines and the pyrimidines bases,bonded in a 3′ to 5′ phosphodiester linkage.

Preferably used oligonucleotides are those modified in either backbone,internucleoside linkages or bases, as is broadly described hereinunder.

Specific examples of preferred oligonucleotides useful according to thisaspect of the present invention include oligonucleotides containingmodified backbones or non-natural internucleoside linkages.Oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone, as disclosed in U.S. Pat. Nos.4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms can also be used.

Alternatively, modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts, as disclosed in U.S. Pat. Nos. 5,034,506;5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562;5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240;5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;5,677,437; and 5,677,439.

Other oligonucleotides which can be used according to the presentinvention, are those modified in both sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for complementation with theappropriate polynucleotide target. An example for such anoligonucleotide mimetic, includes peptide nucleic acid (PNA). A PNAoligonucleotide refers to an oligonucleotide where the sugar-backbone isreplaced with an amide containing backbone, in particular anaminoethylglycine backbone. The bases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Other backbone modifications, which can be used in thepresent invention are disclosed in U.S. Pat. No. 6,303,374.

Oligonucleotides of the present invention may also include basemodifications or substitutions. As used herein, “unmodified” or“natural” bases include the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified bases include but are not limited to other synthetic andnatural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Further bases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Such bases areparticularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. [Sanghvi Y S et al. (1993) AntisenseResearch and Applications, CRC Press, Boca Raton 276-278] and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

As is mentioned hereinabove the agent according to this aspect of thepresent invention can be also a molecule, which indirectly causesreduction of SR activity or expression.

An example of such an agent is a molecule which promotes specificimmunogenic response to a scavenger receptor or an effector thereof inthe subject. Such a molecule can be an SR-BI protein, a fragment derivedtherefrom or a nucleic acid sequence encoding same (see Examples 1-5 ofthe Examples section which follows). Although such a molecule can beprovided to the subject per se, the agent is preferably administeredwith an immunostimulant in an immunogenic composition.

An immunostimulant may be any substance that enhances or potentiates animmune response (antibody and/or cell-mediated) to an exogenous antigen.Examples of immunostimulants include adjuvants, biodegradablemicrospheres (e.g., polylactic galactide) and liposomes into which thecompound is incorporated (see e.g., U.S. Pat. No. 4,235,877).

Vaccine preparation is generally described in, for example, M. F. Powelland M. J. Newman, eds., “Vaccine Design (the subunit and adjuvantapproach),” Plenum Press (NY, 1995).

Illustrative immunogenic compositions may contain DNA encoding ascavenger receptor, such that the protein is generated in situ. The DNAmay be present within any of a variety of delivery systems known tothose of ordinary skill in the art, including nucleic acid expressionsystems, bacteria and viral expression systems. Gene delivery techniquesare well known in the art, such as those described by Rolland, Crit.Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and references citedtherein.

Preferably, the DNA is introduced using a viral expression system (e.g.,vaccinia or other pox virus, retrovirus, lentivirus or adenovirus),which may involve the use of a non-pathogenic (defective), replicationcompetent virus. Suitable systems are disclosed, for example, inFisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexneret al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al.,Science 252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA91:215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; andGuzman et al., Cir. Res. 73:1202-1207, 1993.

Techniques for incorporating DNA into such expression systems are wellknown to those of ordinary skill in the art. The DNA may also be“naked,” as described, for example, in Ulmer et al., Science259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993.The uptake of naked DNA may be increased by coating the DNA ontobiodegradable beads, which are efficiently transported into the cells.

It will be appreciated that an immunogenic composition may comprise botha polynucleotide and a polypeptide component. Such immunogeniccompositions may provide for an enhanced immune response.

Any of a variety of immunostimulants may be employed in the immunogeniccompositions of this invention. For example, an adjuvant may beincluded. Most adjuvants contain a substance designed to protect theantigen from rapid catabolism, such as aluminum hydroxide or mineraloil, and a stimulator of immune responses, such as lipid A, Bortadellapertussis or Mycobacterium tuberculosis derived proteins. Suitableadjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminumhydroxide gel (alum) or aluminum phosphate; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A andquil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may alsobe used as adjuvants.

The adjuvant composition may be designed to induce an immune responsepredominantly of the Th1 type. High levels of Th1-type cytokines (e.g.,IFN-γ, TNF-α, IL-2 and IL-12) tend to favor the induction of cellmediated immune responses to an administered antigen. In contrast, highlevels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend tofavor the induction of humoral immune responses. Following applicationof an immunogenic composition as provided herein, the subject willsupport an immune response that includes Th1- and Th2-type responses.The levels of these cytokines may be readily assessed using standardassays. For a review of the families of cytokines, see Mosmann andCoffinan, Ann. Rev. Immunol. 7:145-173, 1989.

Preferred adjuvants for use in eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), togetherwith an aluminum salt. MPL adjuvants are available from CorixaCorporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc.,Framingham, Mass.), which may be used alone or in combination with otheradjuvants. For example, an enhanced system involves the combination of amonophosphoryl lipid A and saponin derivative, such as the combinationof QS21 and 3D-MPL as described in WO 94/00153, or a less reactogeniccomposition where the QS21 is quenched with cholesterol, as described inWO 96/33739. Other preferred formulations comprise an oil-in-wateremulsion and tocopherol. A particularly potent adjuvant formulationinvolving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion isdescribed in WO 95/17210.

Other preferred adjuvants include Montanide ISA 720 (Seppic, France),SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), theSBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available fromSmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton,Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkylglucosaminide 4-phosphates (AGPs), such as those described in pendingU.S. patent application Ser. No. 08/853,826 and Ser. No. 09/074,720.

A delivery vehicle can be employed with the immunogenic composition ofthe present invention in order to facilitate production of anantigen-specific immune response that targets tumor cells. Deliveryvehicles include antigen presenting cells (APCs), such as dendriticcells, macrophages, B cells, monocytes and other cells that may beengineered to be efficient APCs. Such cells may be genetically modifiedto increase the capacity for presenting the antigen, to improveactivation and/or maintenance of the T cell response, to have anti-tumoreffects per se and/or to be immunologically compatible with the receiver(i.e., matched HLA haplotype). APCs may generally be isolated from anyof a variety of biological fluids and organs, including tumor andperitumoral tissues, and may be autologous, allogeneic, syngeneic orxenogeneic cells.

Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmernan and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaive T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within an immunogenic composition (seeZitvogel et al., Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNF-α to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNF-α, CD40 ligand, LPS, flt3 ligandand/or other compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are categorized as “immature” and “mature” cells, whichallows a simple way to discriminate between two well characterizedphenotypes. Immature dendritic cells are characterized as APC with ahigh capacity for antigen uptake and processing, which correlates withthe high expression of Fcy receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide encoding a SR,such that SR-BI, or an immunogenic portion thereof, is expressed on thecell surface. Such transfection may take place ex vivo, and acomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to the subject, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the SR polypeptide, DNA (nakedor within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule) such as described above. Alternatively, a dendriticcell may be pulsed with a non-conjugated immunological partner,separately or in the presence of the polypeptide.

It will be appreciated that selection of agents which are capable ofreducing the activity or expression of a scavenger receptor or effectorsthereof is preferably effected by examining their effect on at least oneof the above-described scavenger receptor activities. Preferablymodulation of inflammatory cytokine expression in macrophages, such asdescribed in Example 6 of the Examples section.

The above-described agents for reducing expression or activity of ascavenger receptor or of effectors thereof (i.e., active ingredients)can be provided to the subject per se, or as part of a pharmaceuticalcomposition where they are mixed with a pharmaceutically acceptablecarrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the preparationaccountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases. One of the ingredients included in thepharmaceutically acceptable carrier can be for example polyethyleneglycol (PEG), a biocompatible polymer with a wide range of solubility inboth organic and aqueous media (Mutter et al. (1979).

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer a preparation in a local rather thansystemic manner, for example, via injection of the preparation directlyinto a specific region of a patient's body.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, for oralingestion by a patient. Pharmacological preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use. The preparation of the present invention mayalso be formulated in rectal compositions such as suppositories orretention enemas, using, e.g., conventional suppository bases such ascocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro assays. For example, a dose can be formulated in animal modelsand such information can be used to more accurately determine usefuldoses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p.1).

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions including the preparation of the present inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition.

Pharmaceutical compositions of the present invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert.

A number of diseases and conditions, which typically cause inflammatoryresponse in individuals can be treated using the methodology describedhereinabove. Examples of such diseases and conditions are summarizedinfra.

Inflammatory diseases—Include, but are not limited to, chronicinflammatory diseases and acute inflammatory diseases.

Inflammatory Diseases Associated with Hypersensitivity

Examples of hypersensitivity include, but are not limited to, Type Ihypersensitivity, Type II hypersensitivity, Type III hypersensitivity,Type IV hypersensitivity, immediate hypersensitivity, antibody mediatedhypersensitivity, immune complex mediated hypersensitivity, T lymphocytemediated hypersensitivity and DTH.

Type I or immediate hypersensitivity, such as asthma.

Type II hypersensitivity include, but are not limited to, rheumatoiddiseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V.et al., Histol Histopathol 2000 July; 15 (3):791), spondylitis,ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3):189), systemic diseases, systemic autoimmune diseases, systemic lupuserythematosus (Erikson J. et al., Immunol Res 1998; 17 (1-2):49),sclerosis, systemic sclerosis (Renaudineau Y. et al., Clin Diagn LabImmunol. 1999 March; 6 (2):156); Chan O T. et al., Immunol Rev 1999June; 169:107), glandular diseases, glandular autoimmune diseases,pancreatic autoimmune diseases, diabetes, Type I diabetes (Zimmet P.Diabetes Res Clin Pract 1996 October; 34 Suppl:S125), thyroid diseases,autoimmune thyroid diseases, Graves' disease (Orgiazzi J. EndocrinolMetab Clin North Am 2000 June; 29 (2):339), thyroiditis, spontaneousautoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec.15; 165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al., NipponRinsho 1999 August; 57 (8):1810), myxedema, idiopathic myxedema (MitsumaT. Nippon Rinsho. 1999 August; 57 (8):1759); autoimmune reproductivediseases, ovarian diseases, ovarian autoimmunity (Garza K M. et al., JReprod Immunol 1998 February; 37 (2):87), autoimmune anti-sperminfertility (Diekman A B. et al., Am J Reprod Immunol. 2000 March; 43(3):134), repeated fetal loss (Tincani A. et al., Lupus 1998; 7 Suppl2:S107-9), neurodegenerative diseases, neurological diseases,neurological autoimmune diseases, multiple sclerosis (Cross A H. et al.,J Neuroimmunol 2001 Jan. 1; 112 (1-2):1), Alzheimer's disease (Oron L.et al., J Neural Transm Suppl. 1997; 49:77), myasthenia gravis (InfanteA J. And Kraig E, Int Rev Immunol 1999; 18 (1-2):83), motor neuropathies(Kornberg A J. J Clin Neurosci. 2000 May; 7 (3):191), Guillain-Barresyndrome, neuropathies and autoimmune neuropathies (Kusunoki S. Am J MedSci. 2000 April; 319 (4):234), myasthenic diseases, Lambert-Eatonmyasthenic syndrome (Takamori M. Am J Med Sci. 2000 April; 319 (4):204),paraneoplastic neurological diseases, cerebellar atrophy, paraneoplasticcerebellar atrophy, non-paraneoplastic stiff man syndrome, cerebellaratrophies, progressive cerebellar atrophies, encephalitis, Rasmussen'sencephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles dela Tourette syndrome, polyendocrinopathies, autoimmunepolyendocrinopathies (Antoine J C. and Honnorat J. Rev Neurol (Paris)2000 January; 156 (1):23); neuropathies, dysimmune neuropathies(Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl1999; 50:419); neuromyotonia, acquired neuromyotonia, arthrogryposismultiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13;841:482), cardiovascular diseases, cardiovascular autoimmune diseases,atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl 2:S135),myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2:S132),thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9),granulomatosis, Wegener's granulomatosis, arteritis, Takayasu'sarteritis and Kawasaki syndrome (Praprotnik S. et al., Wien KlinWochenschr 2000 Aug. 25; 112 (15-16):660); anti-factor VIII autoimmunedisease (Lacroix-Desmazes S. et al., Semin Thromb Hemost. 2000; 26(2):157); vasculitises, necrotizing small vessel vasculitises,microscopic polyangiitis, Churg and Strauss syndrome,glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis,crescentic glomerulonephritis (Noel L H. Ann Med Interne (Paris). 2000May; 151 (3):178); antiphospholipid syndrome (Flamholz R. et al., J ClinApheresis 1999; 14 (4):171); heart failure, agonist-like β-adrenoceptorantibodies in heart failure (Wallukat G. et al., Am J Cardiol. 1999 Jun.17; 83 (12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int.1999 April-June; 14 (2):114); hemolytic anemia, autoimmune hemolyticanemia (Efremov D G. et al., Leuk Lymphoma 1998 January; 28 (3-4):285),gastrointestinal diseases, autoimmune diseases of the gastrointestinaltract, intestinal diseases, chronic inflammatory intestinal disease(Garcia Herola A. et al., Gastroenterol Hepatol. 2000 January; 23(1):16), celiac disease (Landau Y E. and Shoenfeld Y. Harefuah 2000 Jan.16; 138 (2):122), autoimmune diseases of the musculature, myositis,autoimmune myositis, Sjogren's syndrome (Feist E. et al., Int ArchAllergy Immunol 2000 September; 123 (1):92); smooth muscle autoimmunedisease (Zauli D. et al., Biomed Pharmacother 1999 June; 53 (5-6):234),hepatic diseases, hepatic autoimmune diseases, autoimmune hepatitis(Manns M P. J Hepatol 2000 August; 33 (2):326) and primary biliarycirrhosis (Strassburg C P. et al., Eur J Gastroenterol Hepatol. 1999June; 11 (6):595).

Type IV or T cell mediated hypersensitivity, include, but are notlimited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevittH O. Proc Natl Acad Sci USA 1994 Jan. 18; 91 (2):437), systemicdiseases, systemic autoimmune diseases, systemic lupus erythematosus(Datta S K., Lupus 1998; 7 (9):591), glandular diseases, glandularautoimmune diseases, pancreatic diseases, pancreatic autoimmunediseases, Type 1 diabetes (Castano L. and Eisenbarth G S. Ann. Rev.Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves'disease (Sakata S. et al., Mol Cell Endocrinol 1993 March; 92 (1):77);ovarian diseases (Garza K M. et al., J Reprod Immunol 1998 February; 37(2):87), prostatitis, autoimmune prostatitis (Alexander R B. et al.,Urology 1997 December; 50 (6):893), polyglandular syndrome, autoimmunepolyglandular syndrome, Type I autoimmune polyglandular syndrome (HaraT. et al., Blood. 1991 Mar. 1; 77 (5):1127), neurological diseases,autoimmune neurological diseases, multiple sclerosis, neuritis, opticneuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May;57 (5):544), myasthenia gravis (Oshima M. et al., Eur J Immunol 1990December; 20 (12):2563), stiff-man syndrome (Hiemstra H S. et al., ProcNatl Acad Sci USA 2001 Mar. 27; 98 (7):3988), cardiovascular diseases,cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al., J ClinInvest 1996 Oct. 15; 98 (8):1709), autoimmune thrombocytopenic purpura(Semple J W. et al., Blood 1996 May 15; 87 (10):4245), anti-helper Tlymphocyte autoimmunity (Caporossi A P. et al., Viral Immunol 1998; 11(1):9), hemolytic anemia (Sallah S. et al., Ann Hematol 1997 March; 74(3):139), hepatic diseases, hepatic autoimmune diseases, hepatitis,chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol1990 March; 54 (3):382), biliary cirrhosis, primary biliary cirrhosis(Jones D E. Clin Sci (Colch) 1996 November; 91 (5):551), nephricdiseases, nephric autoimmune diseases, nephritis, interstitial nephritis(Kelly C J. J Am Soc Nephrol 1990 August; 1 (2):140), connective tissuediseases, ear diseases, autoimmune connective tissue diseases,autoimmune ear disease (Yoo T J. et al., Cell Immunol 1994 August; 157(1):249), disease of the inner ear (Gloddek B. et al., Ann N Y Acad Sci1997 Dec. 29; 830:266), skin diseases, cutaneous diseases, dermaldiseases, bullous skin diseases, pemphigus vulgaris, bullous pemphigoidand pemphigus foliaceus.

Examples of delayed type hypersensitivity include, but are not limitedto, contact dermatitis and drug eruption.

Examples of types of T lymphocyte mediating hypersensitivity include,but are not limited to, helper T lymphocytes and cytotoxic Tlymphocytes.

Examples of helper T lymphocyte-mediated hypersensitivity include, butare not limited to, T_(h)1 lymphocyte mediated hypersensitivity andT_(h)2 lymphocyte mediated hypersensitivity.

Autoimmune Diseases

Include, but are not limited to, cardiovascular diseases, rheumatoiddiseases, glandular diseases, gastrointestinal diseases, cutaneousdiseases, hepatic diseases, neurological diseases, muscular diseases,nephric diseases, diseases related to reproduction, connective tissuediseases and systemic diseases.

Examples of autoimmune cardiovascular diseases include, but are notlimited to atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl2:S135), myocardial infarction (Vaarala 0. Lupus. 1998; 7 Suppl 2:S132),thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9), Wegener'sgranulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S.et al., Wien Klin Wochenschr 2000 Aug. 25; 112 (15-16):660), anti-factorVIII autoimmune disease (Lacroix-Desmazes S. et al., Semin ThrombHemost. 2000; 26 (2):157), necrotizing small vessel vasculitis,microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focalnecrotizing and crescentic glomerulonephritis (Noel L H. Ann Med Interne(Paris). 2000 May; 151 (3):178), antiphospholipid syndrome (Flamholz R.et al., J Clin Apheresis 1999; 14 (4):171), antibody-induced heartfailure (Wallukat G. et al., Am J Cardiol. 1999 Jun. 17; 83 (12A):75H),thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 April-June;14 (2):114; Semple J W. et al., Blood 1996 May 15; 87 (10):4245),autoimmune hemolytic anemia (Efremov D G. et al., Leuk Lymphoma 1998January; 28 (3-4):285; Sallah S. et al., Ann Hematol 1997 March; 74(3):139), cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al.,J Clin Invest 1996 Oct. 15; 98 (8):1709) and anti-helper T lymphocyteautoimmunity (Caporossi A P. et al., Viral Immunol 1998; 11 (1):9).

Examples of autoimmune rheumatoid diseases include, but are not limitedto rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 July;15 (3):791; Tisch R, McDevitt H O. Proc Natl Acad Sci units S A 1994Jan. 18; 91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al.,Arthritis Res 2001; 3 (3): 189).

Examples of autoimmune glandular diseases include, but are not limitedto, pancreatic disease, Type I diabetes, thyroid disease, Graves'disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto'sthyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmuneanti-sperm infertility, autoimmune prostatitis and Type I autoimmunepolyglandular syndrome. diseases include, but are not limited toautoimmune diseases of the pancreas, Type 1 diabetes (Castano L. andEisenbarth G S. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res ClinPract 1996 October; 34 Suppl:S125), autoimmune thyroid diseases, Graves'disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 June; 29(2):339; Sakata S. et al., Mol Cell Endocrinol 1993 March; 92 (1):77),spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol2000 Dec. 15; 165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al.,Nippon Rinsho 1999 August; 57 (8):1810), idiopathic myxedema (Mitsuma T.Nippon Rinsho. 1999 August; 57 (8):1759), ovarian autoimmunity (Garza KM. et al., J Reprod Immunol 1998 February; 37 (2):87), autoimmuneanti-sperm infertility (Diekman A B. et al., Am J Reprod Immunol. 2000March; 43 (3):134), autoimmune prostatitis (Alexander R B. et al.,Urology 1997 December; 50 (6):893) and Type I autoimmune polyglandularsyndrome (Hara T. et al., Blood. 1991 Mar. 1; 77 (5):1127).

Examples of autoimmune gastrointestinal diseases include, but are notlimited to, chronic inflammatory intestinal diseases (Garcia Herola A.et al., Gastroenterol Hepatol. 2000 January; 23 (1):16), celiac disease(Landau Y E. and Shoenfeld Y. Harefuah 2000 Jan. 16; 138 (2):122),colitis, ileitis and Crohn's disease.

Examples of autoimmune cutaneous diseases include, but are not limitedto, autoimmune bullous skin diseases, such as, but are not limited to,pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

Examples of autoimmune hepatic diseases include, but are not limited to,hepatitis, autoimmune chronic active hepatitis (Franco A. et al., ClinImmunol Immunopathol 1990 March; 54 (3):382), primary biliary cirrhosis(Jones D E. Clin Sci (Colch) 1996 November; 91 (5):551; Strassburg C P.et al., Eur J Gastroenterol Hepatol. 1999 June; 11 (6):595) andautoimmune hepatitis (Manns M P. J Hepatol 2000 August; 33 (2):326).

Examples of autoimmune neurological diseases include, but are notlimited to, multiple sclerosis (Cross A H. et al., J Neuroimmunol 2001Jan. 1; 112 (1-2):1), Alzheimer's disease (Oron L. et al., J NeuralTransm Suppl. 1997; 49:77), myasthenia gravis (Infante A J. And Kraig E,Int Rev Immunol 1999; 18 (1-2):83; Oshima M. et al., Eur J Immunol 1990December; 20 (12):2563), neuropathies, motor neuropathies (Kornberg A J.J Clin Neurosci. 2000 May; 7 (3):191); Guillain-Barre syndrome andautoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 April; 319(4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. AmJ Med Sci. 2000 April; 319 (4):204); paraneoplastic neurologicaldiseases, cerebellar atrophy, paraneoplastic cerebellar atrophy andstiff-man syndrome (Hiemstra H S. et al., Proc Natl Acad Sci units S A2001 Mar. 27; 98 (7):3988); non-paraneoplastic stiff man syndrome,progressive cerebellar atrophies, encephalitis, Rasmussen'sencephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles dela Tourette syndrome and autoimmune polyendocrinopathies (Antoine J C.and Honnorat J. Rev Neurol (Paris) 2000 January; 156 (1):23); dysimmuneneuropathies (Nobile-Orazio E. et al., Electroencephalogr ClinNeurophysiol Suppl 1999; 50:419); acquired neuromyotonia, arthrogryposismultiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13;841:482), neuritis, optic neuritis (Soderstrom M. et al., J NeurolNeurosurg Psychiatry 1994 May; 57 (5):544) and neurodegenerativediseases.

Examples of autoimmune muscular diseases include, but are not limitedto, myositis, autoimmune myositis and primary Sjogren's syndrome (FeistE. et al., Int Arch Allergy Immunol 2000 September; 123 (1):92) andsmooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother1999 June; 53 (5-6):234).

Examples of autoimmune nephric diseases include, but are not limited to,nephritis and autoimmune interstitial nephritis (Kelly C J. J Am SocNephrol 1990 August; 1 (2):140).

Examples of autoimmune diseases related to reproduction include, but arenot limited to, repeated fetal loss (Tincani A. et al., Lupus 1998; 7Suppl 2:S107-9).

Examples of autoimmune connective tissue diseases include, but are notlimited to, ear diseases, autoimmune ear diseases (Yoo T J. et al., CellImmunol 1994 August; 157 (1):249) and autoimmune diseases of the innerear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec. 29; 830:266).

Examples of autoimmune systemic diseases include, but are not limitedto, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998;17 (1-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin DiagnLab Immunol. 1999 March; 6 (2):156); Chan O T. et al., Immunol Rev 1999June; 169:107).

Infectious Diseases

Examples of infectious diseases include, but are not limited to, chronicinfectious diseases, subacute infectious diseases, acute infectiousdiseases, viral diseases, bacterial diseases, protozoan diseases,parasitic diseases, fungal diseases, mycoplasma diseases and priondiseases.

Graft Rejection Diseases

Examples of diseases associated with transplantation of a graft include,but are not limited to, graft rejection, chronic graft rejection,subacute graft rejection, hyperacute graft rejection, acute graftrejection and graft versus host disease.

Allergic Diseases

Examples of allergic diseases include, but are not limited to, asthma,hives, urticaria, pollen allergy, dust mite allergy, venom allergy,cosmetics allergy, latex allergy, chemical allergy, drug allergy, insectbite allergy, animal dander allergy, stinging plant allergy, poison ivyallergy and food allergy.

Cancerous Diseases

Examples of cancer include but are not limited to carcinoma, lymphoma,blastoma, sarcoma, and leukemia. Particular examples of cancerousdiseases but are not limited to: Myeloid leukemia such as Chronicmyelogenous leukemia. Acute myelogenous leukemia with maturation. Acutepromyelocytic leukemia, Acute nonlymphocytic leukemia with increasedbasophils, Acute monocytic leukemia. Acute myelomonocytic leukemia witheosinophilia; Malignant lymphoma, such as Birkitt's Non-Hodgkin's;Lymphoctyic leukemia, such as Acute lymphoblastic leukemia. Chroniclymphocytic leukemia; Myeloproliferative diseases, such as Solid tumorsBenign Meningioma, Mixed tumors of salivary gland, Colonic adenomas;Adenocarcinomas, such as Small cell lung cancer, Kidney, Uterus,Prostate, Bladder, Ovary, Colon, Sarcomas, Liposarcoma, myxoid, Synovialsarcoma, Rhabdomyosarcoma (alveolar), Extraskeletel myxoidchonodrosarcoma, Ewing's tumor; other include Testicular and ovariandysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignantmelanoma, Mesothelioma, breast, skin, prostate, and ovarian.

In addition to therapeutic advances pioneered by the present invention,the unprecedented findings that antibodies to scavenger receptor areexpressed during an inflammation response may be also employed indiagnostic applications (see Examples 1 of the Examples section whichfollows).

Thus, according to another aspect of the present invention there isprovided a method of diagnosing predisposition to, or presence of, aninflammatory response or diseases related therewith (such as describedabove) in a subject.

As used herein the term “diagnosing” refers to classifying a disease ora symptom as an inflammatory disease, determining a severity of such adisease, monitoring disease progression, forecasting an outcome of adisease and/or prospects of recovery.

The method is effected by detecting autoantibodies to a scavengermolecule in a biological sample obtained from the subject, wherein alevel of the autoantibodies above a predetermined threshold (i.e., thelevel of the same in a biological sample obtained from a healthyindividual) in indicative of the disease in the subject.

As used herein a “biological sample” refers to an antibody-containingsample of cell, tissue or fluid derived from the subject. Antibodiespresent in the sample are typically found within cytoplasmicmembrane-bound compartments (e.g., endoplasmic reticulum and Golgiapparatus) and on the surface of B lymphocytes (which synthesizeantibody molecules) and immune effector cells such as, mononuclearphagocytes, natural killer (NK) cells and mast cells, which expressspecific receptors for binding antibody molecules. Antibodies are alsopresent in the plasma (i.e., fluid portion) of the blood and in theinterstitial fluid of the tissues. Antibodies can also be found insecretory fluids such as mucus, synovial fluid, sperm and milk intowhich certain types of antibody molecules are specifically transported.

Procedures for obtaining biological samples (i.e., biopsying) fromindividuals are well known in the art. Such procedures include, but arenot limited to, blood sampling, joint fluid biopsy, cerebrospinal biopsyand lymph node biopsy. These and other procedures for obtaining tissueor fluid biopsies are described in details in the HealthAtoZ website.

Regardless of the procedure employed, once the biological sample (i.e.,normal and abnormal) is obtained, the titer (number) of antibodymolecules for a scavenger receptor in the biological sample isdetermined.

Antibody titer can determined by techniques which are well known in theart such as ELISA and dot blot using an immobilized antigen (see forExample Abbas, Lichtman and Pober “Cellular and Molecular Immunology”.W.B. Saunders International Edition 1994 pages 56-59). Specifically, theantigen is preferably immobilized on a solid support. To avoidnon-specific binding of antibodies, the solid support is preferablycoated with a nonantigenic protein as well. A peptide is typicallyimmobilized on a solid matrix by adsorption from an aqueous medium,although other modes of immobilization applicable to proteins andpeptides well known to those skilled in the art can be used. Usefulsolid matrices are also well known in the art. Such materials are waterinsoluble and include cross-linked dextran (e.g., SEPHADEX™, PharmaciaFine Chemicals, Piscataway, N.J.), agarose, polystyrene beads about 1 μmto about 5 mm in diameter, polyvinyl chloride, polystyrene, cross-linkedpolyacrylamide, nitrocellulose- or nylon-based webs such as sheets,strips or paddles; or tubes, plates or the wells of a microtiter platesuch as those made from polystyrene or polyvinylchloride.

Once the antigens are immobilized antibody-containing samples are addedin serial dilutions until binding can no longer be observed. Theantibody containing samples can be either a crude sample orimmunoglobulin purified samples (e.g., ammonium sulfate precipitatedfraction and/or chromatography isolated). Immunocomplexes are allowed toform and the support is washed to remove non-specifically boundantisera. Detection of immunocomplexes can be effected by adding labeledantibody-binding molecules such as staphylococcal protein A. The labelcan be an enzyme such as horseradish peroxidase (HRP), glucose oxidase,or the like. In cases where the major indicating group is an enzyme suchas HRP or glucose oxidase, additional reagents are required to indicatethat an immunocomplex has formed. Such additional reagents for HRPinclude hydrogen peroxide and an oxidation dye precursor such asdiaminobenzidine. An additional reagent useful with glucose oxidase is2,2,-azino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).

Radioactive labels may also be used in accordance with the presentinvention. An exemplary radiolabeling agent is a radioactive elementthat produces γ ray emissions, such as ¹²⁵I. Methods of protein labelingare well-known in the art and described in details by Galfre et al.,Meth. Enzyol., 73:3-46 (1981). The techniques of protein conjugation orcoupling through activated functional groups are also applicable. See,for example, Aurameas et al., Scand. J. Immunol., 8(7):7-23 (1978);Rodwell et al., Biotech., 3:889-894 (1984); and U.S. Pat. No. 4,493,795.

Agents of the present invention (described above) can be included in adiagnostic or therapeutic kit. Thus, for example, antibodies and/orchemicals can be packaged in a one or more containers with appropriatebuffers and preservatives and used for diagnosis or for directingtherapeutic treatment.

Preferably, the containers include a label. Suitable containers include,for example, bottles, vials, syringes, and test tubes. The containersmay be formed from a variety of materials such as glass or plastic.

In addition, other additives such as stabilizers, buffers, blockers andthe like may also be added.

The kit can also include instructions for determining if the testedsubject is suffering from, or is at risk of developing inflammation.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 SR-B1 Vaccine Suppresses EAE Disease Progression

The effect of SR-B1 vaccination prior to EAE induction in Lewis rats wasevaluated by manifestation of EAE clinical symptoms. The produced SR-B1autoantibodies were used to evaluate the effect of these antibodies onan ongoing disease.

Materials and Methods

Animals:

Female Lewis rats, approximately six weeks old, were purchased fromHarlan (Jerusalem, Israel) and maintained under SPF (specificpathogen-free) conditions in the Technion animal facility (BruceRappaport Faculty of Medicine, Technion, Haifa, Israel).

Peptide Antigens:

Myelin Basic Protein (MBP) p68-86, Myelin Oligodendrocyte Glycoprotein(MOG) p33-55 were all synthesized on a MilliGen 9050 peptide synthesizerby standard 9-fluorenylmethoxycarbonyl chemistry and purified by highperformance liquid chromatography. Sequence was confirmed by amino acidanalysis and the correct mass was checked by mass spectroscopy. Peptideswith over 95% purity were used.

Immunizations, Active Disease Induction and Disease Score:

Active induction of EAE in each experimental model was done as describedbefore (20, 24, 25). Animals were then monitored for clinical signsdaily by an observer blind to the treatment protocol. EAE was scored asfollows: 0, clinically normal; 1, flaccid tail; 2, hind limb paralysis;3, total hind limb paralysis, accompanied by an apparent front limbparalysis; 4, total hind limb and front limb paralysis.

DNA Vaccines:

A plasmid DNA vaccine-encoding SR-B1 was prepared as described before(10). In brief, RNA extracted from EAE brains of Lewis rats wassubjected to RT-PCR using oligonucleotide primers [sense5′-CCATGGGCGGCAGCTCCAGGGC-3′ (SEQ ID NO: 1), anti-sense5′-CTACAGCTTGGCTTCTTGCAC-3′ (SEQ ID NO: 2)] complimentary to thepublished sequence of SR-B1 (Accession No: AF071495). This RT-PCRreaction mixture was subjected to an amplification program of 1 min at95° C., 1 min at 55° C. and 1 min at 72° C. for 25 cycles. The 1.53 kbamplified product (SEQ ID NO:3) comprising nucleotides 10-1539 of SRB1Rfrom Rattus norvegicus (Accession No: AF071495), was loaded onto a 5%polyacrylamide gel in TAE buffer, gel purified, sequenced and thenligated into a pcDNA3 plasmid. Prior to its use as a vaccine, theconstruct was injected to tibia muscle of rats that were sacrificed atdifferent time points and the expression of RNA encoding SR-B1 wasverified. Under the working condition of the present study, SR-B1 washighly transcribed in the leg muscle for not more than 25 days. DNAvaccines were administrated at a dose of 100 μg plasmid in 100 μl PBS tothe to tibia muscle.

Production and Purification of Recombinant SR-B1:

PCR product of SEQ ID NO: 3 was re-cloned into a PQE expression vector(Qaigen, Chatsworth, Calif.), expressed in E. coli and then purified byan NI-NTA-supper flow affinity purification of 6xHis proteins (Qaigen,Chatsworth, Calif.). After purification, the purity of recombinant SR-B1was verified by gel electrophoresis followed by N-terminus sequencing(Protein Services Unit of the Technion, Haifa, Israel).

Evaluation of Anti SR-B1 Antibody Titer in Sera of Vaccinated Rats:

A direct ELISA assay has been utilized to determine the anti SR-B1antibody titer in DNA vaccinated rats. ELISA plates (Nunc, Roskilde,Denmark) were coated with 50 ng/well of the recombinant SR-B1 proteinproduced in the present study as described above, Sera from DNAvaccinated rats were added in serial dilutions from 2⁵ to 2³⁰ to wellsthat were, or were not, coated previously with recombinant SR-B1.Calculation of each titer was done by comparing the O.D. measured (405nm) in wells coated with SR-B1 to those not coated with this recombinantgene product. Goat anti-rat alkaline phosphatase conjugated IgGantibodies (Sigma) were used as a labeled antibody. p-NitrophenylPhosphate (p-NPP) (Sigma) was used as a soluble alkaline phosphatasesubstrate. Results were collected by ELISA reader (TECAN Spectra rainbowthermo absorber mini-plate reader). Results of triplicates werecalculated as log₂Ab titer±SE.

Evaluation of Anti β-Actin Antibody Titer in Sera of Vaccinated Rats:

Recombinant soluble β-actin was obtained as described before (11). Inbrief, cDNA encoding rat β-actin (the natural cytoplasmic soluble formof β-actin, GenBank Accession NO: NM_(—)031144) was PCR amplified usingspecific oligonucleotide primers [sense 5′-ATGGATGACGATATCGCTGCGCTC-3′(SEQ ID NO:4); anti-sense 5′-CTACCGGCCAGCCAGACG-3′ (SEQ ID NO:5)].Following cloning and sequence verification, the above cDNA was ligatedinto the pcDNA3 vector to be used as a control DNA vaccine). ELISA testwas conducted as described above, but ELISA plates were coated withsoluble β-actin rather than recombinant SR-B1.

IgG Purification:

IgG was purified as described before (11).

Anti SR-B1 Specific Antibody Purification:

Recombinant rat SR-B1 (5 mg) encoded by SEQ ID NO:3, was bound to a CNBractivated Sepharose Column according to the manufactures instructions(Pharmacia biotech, catalog number 17-0820-01). Anti SR-B1 specificantibodies from sera (IgG fraction) of DNA vaccinated rats were loadedon the column and then eluted by an acidic elution buffer (glycine PH2.5). Isotype of the purified antibody was determined by an ELISA assayin which anti rat IgG1a, IgG2b and IgG1 (Jackson, USA) were used asdetection antibodies. Purified antibody was mostly of the IgG2a Isotype(data not shown).

FACS Analysis:

Murine peritoneal macrophages [obtained as described elsewhere (9)],were activated with LPS (1 μg/ml), washed once in FACS buffer (PBS,0.25% BSA, 0.05% sodium azide), and then incubated for 0.5 hours in FACSbuffer enriched with 1% normal rat serum. Cells were then resuspended(4° C., 0.5 hours) in 96-well U plates (10⁶/well) with 10 μL FACS buffersupplemented with 0.5 μg/ml of IgG purified from rats that weresubjected to SR-B1 DNA plasmid administration (anti SR-B1), IgG purifiedfrom EAE rats treated with β-actin encoding DNA (control IgG) or noantibodies (no Ab). Cells were then washed with FACS buffer three timesand incubated (4° C., 0.5 hours) with 50 μL of FACS buffer supplementedwith Goat anti-rat IgG-FITC (#F6258, 1:10,000 dilution; Sigma ChemicalCo.). After incubation cells were washed with FACS buffer twice andanalyzed in the presence of propidium iodide (PI) using a FACScalibur(Becton Dickinson, Mountain View, Calif., USA). Data was collected for10,000 events and analyzed using a Cell Quest program (BectonDickinson).

Statistical and Graphical Methods:

Significance of differences was examined using Student's t-test. A valueof P<0.05 was considered significant. Mann-Whitney sum of ranks test wasused to evaluate significance of differences in mean of maximal clinicalscore with P<0.05 considered significant.

Results

The Effect of SR-B1 Injections on EAE Development:

In the first set of experiments, Lewis rats were subjected to fourweekly administration of plasmid DNA encoding SR-B1, plasmid DNAencoding β-actin (as positive control), or PBS. Two months after lastimmunization EAE was induced. The rats treated with plasmid DNA encodingrat SR-B1 (opened triangles) developed a significantly reduced form ofdisease versus PBS treated rats (closed squares) (FIG. 1 a, mean maximalscore 3±0.28 Vs 1±0.28. p<0.001). DNA vaccine encoding soluble β-actinhad no effect on disease manifestation (FIG. 1 a, opened squares). Atthe peak of disease anti SR-B1 specific antibody titer was determined inall groups (FIG. 1 b). Control EAE rats displayed a significant titeragainst SR-B1 (FIG. 1 b log₂Ab titer of 10±0.4 Vs 6±02 in naïve rats,p<0.05) that continued to persist till 6 days after recovery, and thenregressed back to background levels (not shown). DNA plasmid encodingSR-B1 dramatically amplified this titer (log₂Ab titer of 22±0.86,p<0.001 as compared to each control group) to provide antibody mediatedprotective immunity (FIG. 1 d). EAE rats did not develop a notableantibody titer to soluble β-actin and DNA plasmid encoding this geneproduct could not breakdown tolerance against self (FIG. 1 b). SR-B1autoantibodies were found capable of specific binding to the recombinantSR-B1 (FIG. 1 b), as well as to the natural form of SR-B1 on activatedmacrophages (FIG. 1 c) and HEK293 line cells transfected with rat SR-B1(not shown). These autoantibodies could also adoptively transfer EAEresistance to other rats (FIG. 1 d, mean maximal score of 3.3±0.3 incontrol rats Vs 1.66±0.18, p<0.01).

Example 2

The Anti-Inflammatory Mechanism of Anti SR-B1 Antibodies

The mechanism of action of anti SR-B1 antibodies was evaluated bytesting their ability to modify cytokine production by murine peritonealmacrophages.

Material and Methods

Murine Peritoneal Macrophages:

Murine peritoneal macrophages were isolated as described before (9).Cells were then activated in vitro with 1 μg/ml LPS for 48 in thepresence of (0, 10, 50 and 100 μg/ml) anti SR-B1 polyclonalautoantibodies (CNBr purified, as described in Example 1 of the Examplessection), or control IgG from normal rat serum (purified as described inExample 1 of the Examples section).

Cytokine Determination:

Levels of IL-12, TNF-α and IL-10 were determined by ELISA (TECAN Spectrarainbow thermo absorber mini-plate reader) using commercially availablekits: mouse IL-12 (R&D system Inc. Minneapolis, Minn.), TNF-α & IL-10(Diaclone, Besancon, France).

Statistical and Graphical Methods:

Statistical methods were as described in Example 1 of the Examplessection.

Results

The Mechanism of Action of Anti SR-B1 Antibodies:

To explore the mechanistic basis by which anti SR-B1 antibodies affectthe function of the immune system, these antibodies were purified andadded to freshly isolated peritoneal macrophages that were activated invitro with LPS. The presence of anti SR-B1 antibodies in the peritonealmacrophages culture effectively suppressed IL-12 and TNF-α production(FIG. 2 a and FIG. 2 b, closed squares) and at the same time inducedIL-10 production (FIG. 2 c, closed squares) as compared to control IgG(FIG. 2, open squares), all in a dose dependent manner (FIG. 2). Thusanti SR-B1 antibodies redirect the polarization of macrophages from apro-inflammatory to anti-inflammatory mediators.

Example 3 SR-B1 Vaccine Suppresses an Ongoing EAE

The effect of SR-B1 vaccine on an ongoing disease was evaluated by EAEC57BL/6 mice vaccination with plasmid DNA encoding SR-B1, 12 days afteronset of the disease.

Materials and Methods

Animals:

C57BL/6 mice, approximately six weeks old, were purchased from Harlan(Jerusalem, Israel) and maintained under SPF conditions in the Technionanimal facility (Bruce Rappaport Faculty of Medicine, Technion, Haifa,Israel).

EAE Induction:

EAE was induced according to Semi-chronic model of MOG induced EAE inthe C57BL/6 mice as described before (20).

DNA Vaccines:

SR-B1 plasmid DNA vaccine was prepared as described in Example 1 of theExamples section.

Mouse Vaccination:

12 days after the induction of EAE (1-2 days after its onset) sick micewere separated into 3 group of 6 C57BL/6 mice that were subjected toeither a single administration of plasmid DNA encoding SR-B1(pcDNA3-SR-B1), a single administration of pcDNA3 (empty vector), or noinjection (control). Vaccination was effected by administration of 100μg plasmid in 100 μl PBS as described Example 1 of the Examples section.

Statistical and Graphical Methods:

Statistical analysis was effected as described in Example 1 of theExamples section.

Results

The Effect of SR-B1 Injections on the Dynamics of an Ongoing EAEDisease:

Semi-chronic model of MOG induced EAE in the C57BL/6 mice (20) was usedto evaluate the effect of a plasmid DNA vaccine encoding SR-B1 on thedynamics of an ongoing EAE disease (FIG. 3). At this time anti SR-B1specific antibody titer excided the level of log₂Ab titer of 11±0.4 (Vs6±0 in naïve mice) this titer accelerated to log₂Ab titer of 21±0.66 inSR-B1 encoding DNA treated mice (p<0.001) (Data not shown). Thisacceleration was followed by a fast entry to remission in treated mice(FIG. 3, p<0.001). These data further suggest that SR-B1 encoding DNAvaccine amplifies a pre-existing regulatory response.

Example 4 Anti SR-B1 Monoclonal Antibodies Suppress Ongoing EAE

Anti SR-B1 monoclonal antibody was produced, and tested for its abilityto modulate cytokine production by peritoneal activated macrophages aswell as to affect an ongoing EAE disease

Materials and Methods

Production of Monoclonal Anti SR-B1 Antibody:

C57/B6 mice were subsequently immunized (3 weekly immunizations) withthe SR-B1 encoding DNA plasmid. Two weeks after the last administration,these mice were subjected to active induction of EAE. Spleen cells wereobtained for production of monoclonal antibodies two weeks later withSP2 cells (ATCC) as a fusion partner as described before (E. Harlow & D.Lane, Antibodies, Cold Spring Harbor Laboratory Press, 1998). Screeningof positive hybridoma was done in two steps of selection. The first oneselected positive antibodies producing cells according to the ability tobind SR-B1 over expressed by HEK293. Supernatant isolated from hybridomaclones (1000 wells) was then subjected to FACS analysis for theirability to bind SR-B1.

Antibodies and Peritoneal Macrophages:

Peritoneal macrophages were obtained from thioglycollate (25%, 3 ml)injected C57BL/6 mice and co-cultured with 1 μg/ml LPS for 48 in thepresence, or absence, of anti SR-B1 polyclonal antibody (1:100,Calbichem, San Diego, Calif.), the monoclonal anti SR-B1 antibodygenerated as described above (Clone 5D8, 10 μg/ml), isotype matchedcontrol antibody (IgG1_(κ), Sigma), HDL (Chemicon InternationalTemecula, Calif.) or anti murine CD36 (5 μg/ml, Santa CruzBiotechnology, Santa Cruz, Calif.). Cytokine levels were determined byELISA as described in Example 2 of the Examples section. Results areshown as mean triplicates ±SE.

Antibodies Administration to EAE Rats:

Lewis rats were subjected to active EAE as described in Example 1 of theExamples section. 1-2 days after the onset of disease these Lewis rats(6 in each group) were treated, every other day, with 100 μg/ml ofmonoclonal antibody 5D8 (anti SR-B1 monoclonal antibody selected fromhybridoma, as described above, which recognizes the murine homolog ofSR-B1), control murine IgG1 (Sigma), anti SR-B1 polyclonal antibody(produced as described in Example 1 of the Examples section), controlrat IgG2a purified from EAE rats that have been subjected to an emptyplasmid DNA vaccination or PBS. Data was obtained by an observer blindto the experimental protocol as described in Example 1 of the Examplessection. Results are presented as mean maximal score ±SE.

Results

The Effect of Monoclonal Antibody to SR-B1 on Cytokine Production of LPSActivated Peritoneal Macrophages:

The ability of anti SR-B1 specific monoclonal antibody to alter thecytokine profile produced by peritoneal macrophages and by the murinemonocyteric cell line J774 (ATCC) was evaluated by their cytokinesexpression. Table 1 compares the competence of one of these antibodies(clone 5D8, IgG1) to decrease TNF-α and increase IL-10 production by LPSactivated murine peritoneal macrophages to the effect of commerciallyavailable polyclonal antibody to SR-B1, to CD36 and to HDL. Eachresponse was optimized during a preliminary experiment in whichdifferent doses were determined for properties to affect theseparameters.

TABLE 1 The effect of anti SR-B1 monoclonal antibody on cytokineproduction of LPS activated peritoneal macrophages Treatment TNF-α(pg/ml) IL-10 (pg/ml) — 3280 ± 210 730 ± 80 Anti SR-B1 (polyclonal) 1760± 190 1240 ± 160 Anti SR-B1 (clone 5D8) 1640 ± 140 1630 ± 150 Controlmurine IgG1_(κ) 3350 ± 260  710 ± 100 HDL (100 μg/ml) 2560 ± 130 146 ±30 Anti CD36 (polyclonal) 4660 ± 330 330 ± 90

The anti SR-B1 monoclonal antibody produced by spleen cells isolatedfrom the mice treated with SR-B1 encoding DNA vaccine and the anti SR-B1specific polyclonal antibody could both suppress TNF-α production(1640±140 and 1760±190 pg/ml Vs 3280±210 pg/ml in control samples,p<0.001, Table 1). Control IgG1_(κ) (Sigma) had no significant effect onthe production of these cytokines (Table 1). Very similar results wereobtained using the murine monocyteric cell line J774 or peritonealmacrophages from rats (data not shown). HDL could reduce TNF-αproduction (2560±130 Vs 3280±210, p<0.05) but also decreased theproduction of the anti-inflammatory cytokine IL-10 (Table 1). Anti CD36antibodies markedly increased TNF-α production (Table 1, p<0.01).

The Effect of Anti SR-B1 Monoclonal Antibody on EAE:

The ability of the isolated anti SR-B1 monoclonal antibody to suppressEAE was evaluated in adoptive transfer experiments. The antibodies weretransferred to EAE rats just after the onset of disease and led to asignificant reduction in disease severity (mean maximal score of1.66±0.2 in treated rats Vs 3±0.3 in control rats, p<0.05, FIG. 4).

Example 5 Anti SR-B1 Antibodies Suppress an Ongoing Inflammatory BowelDisease (IBD)

Anti SR-B1 antibodies were used to treat an induced IBD in Lewis rats.

Materials and Methods

Animals:

Female Lewis rats, approximately six weeks old (120-150 g), weremaintained as described in Example 1 of the Examples section.

Induction of Colitis:

Experimental colitis was induced by intrarectal instillation of 250 μlof 125 mg/ml 2,4,6-trinitrobenzene sulfonic acid (TNBS) solution (Fluka,cat#92822) dissolved in 50% ethanol, using 8 cm neonate feeding tube asdescribed before [Fiorucci, S. et al., Immunity, 17:769., 2002]. 24hours post injection all rats developed bloody diarrhea and severediarrhea in the next day, accompanied with continuous loss of weight.

Anti SR-B1 and IgG Antibodies:

Specific anti SR-B1 polyclonal antibodies and IgG antibodies werepurified as described in Example 1 of the Examples section.

Rat Vaccination:

5 days after the induction of IBD, when the clinical manifestation ofdisease was apparent in all rats, they were separated into 3 groups of 6equally sick rats each. On days 6, 8 and 10 after induction of diseasethese groups were administered with either rat anti SR-B1 polyclonalantibodies (DNA vaccination based antibody, produced as described inExample 1 of the Examples section, 100 μg/rat in PBS), rat IgG frompre-immunized rats (100 μg/rat in PBS), or PBS (control).

Histological Sections:

Histological sections were conducted according to Fiorucci et al.,[Fiorucci, S., et al., Immunity 17:769, 2002]. Briefly, on day 15 postinduction of colitis, Lewis rats were sacrificed and colons (cecum torectum) were extracted, flushed with PBS and fixed in NBF (neutralbuffered formalin). Paraffin sections (6 μm) were made and stained withH&E (hematoxylin and eosin). Each section represents ˜250 sections thatwere screened by an observer blind to the experimental protocol.

Results

Diarrhea Symptoms:

On day 15, post induction of the colitis, all 6 rats treated with antiSR-B1 showed no signs of diarrhea and solid feces could be seen in thecage. Control rats (positive control of PBS treated rats and ratstreated with control IgG) suffered from severe diarrhea with no solidfeces in the cage.

Histological Sections of Rats Colons:

Histological colon sections of untreated rats showed normal mucosa withwell defined colonic crypts and glands. Histological colon sections ofboth control rats (positive control of PBS treated rats and rats treatedwith control IgG) taken 15 days post induction of colitis, showed severesigns of inflammation, megacolon, and signs of perforations (FIGS. 5b-c). Positive control of PBS treated rat sections showed diffuse,mononuclear inflammatory infiltrates in the mucosa and lamina propria,submucosa, and muscularis mucosae (FIG. 5 b). Sections displayed variousdegrees of damage to colonic tissue, ranging from heavily infiltratedareas with epithelial exfoliation to lesions with total destruction ofmucosal surface, transmural infiltration, necrosis, and loss of tissuearchitecture. The control IgG colon sections showed massive destructionof colonic tissue with vast necrotic areas, absence of glandularstructure and complete loss of tissue architecture (FIG. 5 c).Congestion and edema and congestion were seen in and around bloodvessels. Damage was continuous from rectum and extended proximally up tocecum (FIG. 5 c). Histological colon sections of rats treated with antiSR-B1 antibodies showed very little signs of inflammation, noperforations, and smaller and paler colons (FIG. 5 d). Sections showedmoderate inflammatory infiltration, compared to control IgG treated rats(FIG. 5 d Vs. FIG. 5 c). Although infiltrated, colonic mucosa seemsintact with visible brush border and glands and no transmuralinfiltrates. Submucosa was also much less infiltrated with no wallthickening (FIG. 5 d).

These results indicate that treatment of IBD with anti SR-B1 antibodieseffectively reduces diarrhea symptoms through reduction of damage to thecolon.

Example 6 Therapeutic Monoclonal Human Anti SR-B1 Antibody

A monoclonal human anti SR-B1 antibody was produced for therapeutic use.

Materials and Methods

SR-B1 Encoding Plasmids:

DNA encoding human SR-B1 (CLA-I) was amplified using sense primer: 5′CCATGGGCTGCTCCGCCAAA 3′ (SEQ ID NO: 6), and anti-sense primer: 5′CTACAGTTTTGCTTCCTGCAG 3′ (SEQ ID NO: 7) The above described reactionmixture was subjected to an amplification program of 1 min at 95° C., 1min at 55° C. and 1 min at 72° C. for 25 cycles, generating 1.53 kb DNAfragment of SEQ ID NO:8 (Homo sapiens encoding SR-B1 mRNA, nucleotides70-1599 from accession number: Z22555). After PCR reaction, the mixturewas loaded onto a 5% polyacrylamide gel in TAE buffer. PCR product wasgel-purified, cloned into a pUC57/T vector (T-cloning kit K1212; MBIFermentas, Vilnius, Lithuania) and then used to transform E. coli cells.Clones were then sequenced (Sequenase version 2; Upstate Biotechnology,Cleveland, Ohio) and transferred into a pcDNA3 vector (Invitrogen, SanDiego, Calif.). Large-scale preparation of plasmid DNA was conductedusing Mega prep (Qiagen, Chatsworth, Calif.).

Cells:

HEK293 (ATCC) were transfected with human SR-B1 as described before[Scarselli E, et al., EMBO J. 21(19):5017-25, 2002]. Expression wasverified by FACS analysis as described before [Scarselli E, et al., EMBOJ. 21(19):5017-25, 2002].

Production of Monoclonal Human Anti SR-B1 Antibody:

Human anti SR-B1 monoclonal antibodies were produced according to one ofthe two following protocols:

Protocol I

C57/B6 mice were subsequently immunized (3 weekly immunizations) withthe human SR-B1 (SEQ ID NO:8) encoding DNA plasmid. Two weeks after thelast administration, these mice were subjected to active induction ofEAE. Spleen cells were obtained for production of monoclonal antibodiestwo weeks later with SP2 cells (ATCC) as a fusion partner as describedbefore (E. Harlow & D. Lane, Antibodies, Cold Spring Harbor LaboratoryPress, 1998). Screening of positive hybridoma was done in two steps ofselection. The first one selected positive antibodies producing cellsaccording to the ability to bind the recombinant SR-B1 over expressed byHEK293. Supernatant isolated from hybridoma clones (1000 wells) was thensubjected to FACS analysis for their ability to bind SR-B1

Protocol II

The cloned human SR-B1 (SEQ ID NO:8), obtained as described above, wasre-cloned into a pQE expression vector, expressed in E. coli (Qiagen)and then purified by an NI-NTA-supper flow affinity purification of6×His proteins (Qiagen). After purification, the purity of therecombinant human SR-B1 was verified by gel electrophoresis followed bysequencing (N terminus) by the Technion's sequencing services unit(Technion, Haifa, Israel). This recombinant human SR-B1 was theninjected into 10-weeks old BALB/C mice. First immunization was of 50 μgpeptide emulsified in CFA [incomplete Freund's adjuvant (IFA)supplemented with 10 mg/ml heat-killed Mycobacterium tuberculosis H37Rain oil; Difco Laboratories, Detroit, Mich.] at a total volume of 400 μlinto the peritoneal cavity. Later on, in a 3 weeks interval these micewere administrated with 50 μg/400 μl or recombinant human SR-B1emulsified in IFA (Difco Laboratories, Detroit, Mich.). Three weeksafter the third interval mice were injected (intravenous) with 50 μg ofrecombinant human SR-B1 in 100 μl PBS. Three days later spleen cellswere obtained and preparation of monoclonal antibodies was conducted asdescribed above.

ELISA—The indirect ELISA was used to screen hybridomas for antibodiesagainst SR-BI, as follows. Ninety six-well microtiter plates (NUNC) werecoated with 50 ng/ml of immuno (recombinant) SR-BI (SEQ ID NO: 8) inphosphate buffered saline (PBS) overnight at 4° C., followed by blockingwith 200 μl of 5% BSA in PBS. Then 100 μl of hybridoma supernatants wereadded and incubated for 1 hr at room temperature (RT). The plates werewashed 4 times with PBS containing 0.05% Tween 20 (PBS-T), and thensupplemented with peroxidase-conjugated goat anti-mouse IgG antibody for1 hr at RT, and washed 5 times with PBS-T. Then 100 μl of substratesolution 3,3′,5,5′-tetramethyl Benzedrine liquid (ICN biomedical INC,Germany, TMB) were added. The reaction was stopped using 2.5M H₂SO₄ andthe absorbance was read by an ELISA reader at a wavelength of 450 nm andbackground of 630 nm.

Cell binding assay—HEK 293 cell line was stably transfected with pcDNAencoding SR-BI (pcSR-BI). Positive clones were selected using neomycin(G418). The positively isolated ELISA hybridoma clones (isolated asdescribed above) were taken into the second screen. Ninety six welldisposable flexible polyvinyl chloride microtitration plates (Dynatechlaboratories, Virginia) were seeded with 1*10⁶ pcSR-BI-expressing HEK293 cells. The cells were washed twice with PBS before 100 μl ofhybridomas supernatants were added for 30 minutes on ice. Following 3washes with PBS, peroxidase-conjugated goat anti-mouse IgG antibody wasadded for additional 20 min on ice. Following 3 washes with PBS 100 μlof substrate solution (TMB) was added. The reaction was stopped using2.5M H₂SO₄. After a short centrifugation, the reaction was transferredinto a clean well and the absorbance was read by an ELISA reader at awavelength of 450 nm and background of 630 nm.

Example 7 In Vitro Characterization of Anti SR-BI Therapeutic Antibodies

Human SR-B1 cross-reactive antibodies (with CLA-I) generated asdescribed in Example 6 according to protocol 2 were in-vitro screenedand characterized. The most successful antibody obtained was E12, whichwas further in-vitro characterized as further described hereinbelow.

Materials and Experimental Procedures

Immunoblot analysis—For single-label immunohistochemistry, standardmethodology was used whereby sections were incubated with primaryantibodies (1:100), followed by incubation with secondary antibodies(1:100). Mouse IgG and rabbit polyclonal IgG were used as controlantibodies.

Isotype analysis—Isotype analysis was done using Serotec kit (AbDSerotec, Raleigh, N.C., USA).

Culture of peritoneal macrophages—Resident macrophages were obtainedfrom a peritoneal lavage with PBS. Elicited macrophages were harvested 5days following i.p. injection of 3 ml of 3% Thioglycollate (TG, Difco,Livonia, Mich.). Peritoneal exudate cells were washed, re-suspended inRPMI 1640 medium supplemented with 10% FCS, 1% penicillin, 1%streptomycin, and incubated in 24 flat-bottom plates (10⁶ cells per wellin 1 ml) for overnight at 37° C. Nonadherent cells were then removed byvigorous washing (three times), and macrophages monolayers wereincubated for 1-10 days in antibiotic-free RPMI containing 10% FCS.Fresh medium was provided every 3 days.

IL-10 production by macrophage culture The peritoneal macrophagesgenerated as described above were treated with mAb E12 with or without0.5 μg/ml LPS (Sigma) for 24 hr at 37° C. Supernatants from eithertreated or untreated macrophages were assayed for the presence of IL-10or using immunoenzymatic ELISA kits (Biolegend).

Nitrite production by macrophage culture—Nitrite formation measurementwas done according as described by [Katakura, T., M. Miyazaki, M.Kobayashi, D. N. Herndon, and F. Suzuki. (2004). CCL17 and IL-10 aseffectors that enable alternatively activated macrophages to inhibit thegeneration of classically activated macrophages. J Immunol 172:1407].Peritoneal macrophages (1*10⁶/ml) were seeded in 24-well plates asdescribe above. Following treatment with LPS and/or mAb E12 thesupernatant was taken and NO production was assayed by measuring theaccumulation of nitrite in the culture medium by Griess reaction usingGriess reagent system kit (Promega). Briefly: an equal volume of Griessreagent (Sulfanilamide Solution) and macrophage supernatants wasincubated for 10 min at RT in a dark room. An equal volume ofN-1-napthylethylenediamine dihydrochloride (NED) was then added for 10min. An ELISA reader measured the absorbance at 550 nm. Nitriteconcentration was determined using NaNO₂ as a standard.

Results

Isotype analysis of E12 revealed it to be IgG1. The purified E12 wasreacted with a nitrocellulose membrane containing various recombinantproteins. As shown in FIG. 6, mAb E12 cross reacted with SR-BI and CLA-1but not with MIP, CXCL6 or IL-27. These results indicate that theantibody specifically recognizes scavenger B1 receptor in across-species dependent manner and is capable of recognizing thedenatured form of the protein indicating that it is directed against anexposed epitope of the native protein, as further demonstrated by itsability to neutralize SR-B1 activity.

The ability of E12 to neutralize SR-B1 signaling, was in vitro assayedon cultured peritoneal macrophages. As shown in FIG. 7 culturedperitoneal macrophages treated for 24 hours with 0.5 μg/ml LPS and withmAb E12, or with isotype matched control IgG, produced significantlyhigher IL-10 in the presence of increasing amounts of E12 than comparedto control treated cells.

These results were substantiated when following NO levels in thepresence of E12 antibody and LPS (0.5 μg/ml). As shown in FIG. 8, mAbE12 suppressed NO synthesis (as determined by nitrite levels) byperitoneal macrophages in a dose dependent manner. Control matchedisotypes had no effect of NO levels.

The variable regions of E12 heavy chain (VH) and light chain (VK) weresequenced and their CDR composition determined. SEQ ID NO: 9 and 10 showthe amino acid and nucleic acid sequences of framework 1 (FWR1) of E12light chain, respectively. SEQ ID NO: 11 and 12 show the amino acid andnucleic acid sequences of CDR1 of E12 light chain, respectively. SEQ IDNO: 13 and 14 show the amino acid and nucleic acid sequences offramework 2 (FWR2) of E12 light chain, respectively. SEQ ID NO: 15 and16 show the amino acid and nucleic acid sequences of CDR2 of E12 lightchain, respectively. SEQ ID NO: 17 and 18 show the amino acid andnucleic acid sequences of framework 3 (FWR3) of E12 light chain,respectively. SEQ ID NO: 19 and 20 show the amino acid and nucleic acidsequences of CDR3 of E12 light chain, respectively. SEQ ID NO: 21 and 22show the amino acid and nucleic acid sequences of framework 4 (FWR4) ofE12 light chain, respectively.

SEQ ID NO: 23 and 24 show the amino acid and nucleic acid sequences offramework 1 (FWR1) of E12 heavy chain, respectively. SEQ ID NO: 25 and26 show the amino acid and nucleic acid sequences of CDR1 of E12 heavychain, respectively. SEQ ID NO: 27 and 28 show the amino acid andnucleic acid sequences of framework 2 (FWR2) of E12 heavy chain,respectively. SEQ ID NO: 29 and 30 show the amino acid and nucleic acidsequences of CDR2 of E12 heavy chain, respectively. SEQ ID NO: 31 and 32show the amino acid and nucleic acid sequences of framework 3 (FWR3) ofE12 heavy chain, respectively. SEQ ID NO: 33 and 34 show the amino acidand nucleic acid sequences of CDR3 of E12 heavy chain, respectively. SEQID NO: 35 and 36 show the amino acid and nucleic acid sequences offramework 4 (FWR4) of E12 heavy chain, respectively.

Blast analysis of E12 light chain sequence showed no homology to antiSR-B1 antibodies which sequence is available to date. Closest homologywas as follows:

1. A set of closely related antibodies which dominate the primaryantibody response to the antigenic site CB of the A/PR/8/34 influenzavirus hemagglutinin;

2. anti-cocaine monoclonal antibody K1-4 light chain variable region;

3. ANA immunoglobulin kappa light chain [Mus musculus]; and

4. anti-GBM [Anti-Glomerular Antigen Antibody-Producing Cells in theKidneys of MRL/MpJ-Fas(lpr) Mice] immunoglobulin kappa chain variableregion [Mus musculus].

Example 8 A Monoclonal Antibody to SR-B1 is Capable of SuppressingOngoing EAE and IBD

The monoclonal antibody generated as taught in Example 6 above was shownhighly effective in suppressing ongoing EAE and TNBS induced IBD, asfurther described hereinbelow.

Materials and Methods

Induction of EAE in mice and suppression of the ongoing disease with mAbto SR-B1—A group of 18 C57BL/6 mice was subjected to MOGp35-55 inducedEAE. At the onset of disease (day 13) these mice were separated intothree equally sick groups. On this day and on days 15 and 17 thesegroups were intravenously administered with 500 μg E12 mAb, isotypematched human IgG (IgG1), or PBS and followed for clinical manifestationof disease (FIG. 9) by an observer blind to the experimental protocol.

Spinal cord histopathology—Histological examination of H&E-stainedsections of formalin-fixed, paraffin-embedded sections of the lowerthoracic and lumbar regions of the spinal cord was performed. Eachsection was evaluated without knowledge of the treatment status of theanimal. The following scale was used: 0, no mononuclear cellinfiltration; 1, one to five perivascular lesions per section withminimal parenchymal infiltration; 2, five to 10 perivascular lesions persection with parenchymal infiltration; and 3, >10 perivascular lesionsper section with extensive parenchymal infiltration. The meanhistological score ±SE was calculated for each treatment group.

Immunohistochemistry—For single-label immunohistochemistry, standardmethodology was used whereby sections were incubated with primaryantibodies (1:100), followed by incubation with secondary antibodies(1:100). Mouse IgG and rabbit polyclonal IgG were used as controlantibodies.

Induction of experimental Colitis in Lewis rats—See Example 5.

Treatment protocol for antibody transfer—On days 6, 8 and 10 postinduction of experimental colitis, 500 μg of mAb E12 was injectedintravenously via a tail vein. Human IgG1 (Sigma) was used as a controlantibody.

Sample collection—On day 12, the rats were killed underketamine-xylasine anesthesia. The terminal colon was then stripped,gently washed with PBS, opened longitudinally and macroscopicallyevaluated according to a modification of the criteria described byMorris Gut (2004); 53; 99-107. Colonic injury was scored on a 0 (normalcolon) to 5 (severe damage) scale, (see Table 2, below).

Colon Histopathology—Tissues (terminal colon, mesentery lymph nodes andspleens) were fixed in 10% neutral buffered formalin and embedded inparaffin. Hematoxylin and eosin stained sections of the colon wereevaluated histologically for four parameters: extent of ulceration,submucosal infiltration, crypt abscesses and wall thickening (see Table3). The sum of all scores determined a rating of slight to severecolonic inflammation.

Immunohistochemistry—Serial sections from formalin-fixed,paraffin-embedded specimens were deparaffinized and rehydrated indecreasing concentrations of ethyl alcohol. Tissue sections wereincubated with fresh 3% H₂O₂ in methanol for 10 min and then washed withPBS. Sections were then treated by microwave for 15 min in 90° C. incitrate buffer and blocked with 10% donkey serum for 30 min.Immunohistochemical analysis was carried out using primary antibodiesagainst rat IL-10 (polyclonal goat anti rat IL-10, R&D), CD3 (mAb mouseanti rat, Pharmingen) and ED1 (mAb mouse anti rat, Serotec) over nightat 4° C. in a humidified chamber. Biotinylated donkey anti goat or antimouse IgG were used as secondary antibodies, followed by astreptavidin-horseradish peroxidase (Zymed). The reaction was developedusing aminoethylcarbazole substrate kit (Zymed).

TABLE 2 Macroscopic assessment of colonic damage Macroscopic damageScore No damage 0 Hyperemia but no ulcers 1 Fibrosis but no ulcers 2Ulceration/necrosis <1 cm 3 Ulceration/necrosis <2 cm 4Ulceration/necrosis >2 cm 5

TABLE 3 Microscopic assessment of colonic inflammation Histologicalappearance Score Extent of ulceration No ulcer 0 Small ulcers (<3 mm)1-2 Large ulcers (>3 mm) 3-5 Submucosal infiltration None 0 Mild 1Moderate 2-3 Severe 4-5 Crypt abscesses None 0 Rare 1-2 Diffuse 3-5 Wallthickness (μm) <470 0 <600 1 <700 2 <800 3 <900 4 >900 5

Results

Anti SR-BI mAb Suppress Long-Term Ongoing EAE

Three groups of mice models of EAE displaying similar clinicalmanifestations were subjected to monoclonal antibody therapy and controltreatments. As shown in FIG. 9, mice treated with PBS or control IgGcontinued to develop severe EAE, while those treated with the anti SR-BImAb E12 went into fast remission without residual sign of disease (FIG.9).

On day 19, spleen cells were isolated from representative mice of eachgroup and cultured for 72 h with the target antigen with which diseasewas induced. Levels of IL-10, IL-12 (p40 subunit) and IL-4 were thenrecorded using commercially available ELISA kits. FIGS. 10 a-c summarizethe results of this experiment showing a marked elevation in IL-10production (p<0.001), a significant elevation in IL-4 production(p<0.01) accompanied by a significant reduction in IL-12 production(p<0.01). These results are consistent with the in vitro properties ofthis antibody (see FIGS. 10 a-c and may explain, at least in part thebeneficial effect of this therapy (FIG. 9).

Spinal cord (lumbar spinal cord) sections obtained on day 19 fromcontrol EAE mice and from those subjected to IgG1 or E12 therapy (seeFIG. 9) were subjected to an immunohistological analysis of theexpression of SR-BI on leukocytes around high endothelial venules (HEV).FIGS. 11 a-c show representative sections of untreated control EAE mice,EAE mice treated with E12 and EAE mice treated with control IgG1,respectively. In all sections of sick mice leukocytes entering the CNShighly expressed SR-BI. The reduction in the density of these cells inanti SR-BI treated mice could be explained, in part, by the reducednumber of invading leukocytes resulting from the decrease in theinflammatory process (i.e. lower histological score).

Finally representative sections from these groups were subjected toimmunohistological analysis of IL-10, using a commercially availableanti IL-10 mAb. FIGS. 11 d-f show representative sections of untreatedcontrol EAE mice, EAE mice treated with E12 and EAE mice treated withcontrol IgG1, respectively. The elevation in IL-10 production insections of mice treated with E12 is apparent compared to each of thecontrol groups. These results support the above in-vitro results,substantiating the anti-inflammatory role of anti SR-B1 therapy.

Anti SR-BI mAb Suppresses Experimental Colitis

Similar analysis of the effect of anti-SR-B1 monoclonal antibodies onIBD was effected on a rat model of colotis. The following summarizesmacroscopic and microscopic analyses on colitis induced rats (6 rats pergroup), as well as representative samples of histopathological analysis,followed by immunohistochemistry detection of ED1 positive cells(macrophages), CD3⁺ T cells and IL-10 staining in all groups.

Table 4 below clearly shows that a significant reduction in macroscopicand microscopic scores of disease which is accompanied by a markedreduction in histopathological changes in the colon.

TABLE 4 TNBS control TNBS IgG TNBS E12mAb Mean macroscopic    4 ± 0.66 4.2 ± 0.66 2.66 ± 0.5* assessment Mean microscopic 15 ± 2 17.5 ± 2.2 6.5 ± 2** assessment *p < 0.01, **p < 0.001

FIGS. 12 a-e show representative histological colon sections obtained atday 12 of IBD onset from naïve rats (FIG. 12 a), positive control ratssuffering from TNBS induced IBD (FIG. 12 b), rats suffering from TNBSinduced IBD that were subjected to repeated administration of isotypematched control IgG (FIG. 12 c) in comparison to those treated with mAbE12 (FIGS. 12 d-e). As shown structural changes between E12 treatedcolon and control are evident. This may be explained by the shift incytokine profile from pro-inflammatory (in control treated animals) toanti-inflammatory cytokines (in E12 treated animals) as shown in FIGS.13 a-i.

FIGS. 13 a-c show sections of untreated IBD induced rats. Massivesubmucosal infiltration of macrophages (ED1⁺) and both mucosal andsubmucosal infiltration of T cells (CD3+) are shown. IL-10 productionwas barely detected, mainly in the mucosa.

FIGS. 13 d-f show sections of isotype matching control treated animals.Submucosal infiltration of macrophages (ED1+), mucosal infiltration of Tcells (CD3+) and minor IL-10 production in the mucosa are detected.

FIGS. 13 g-i show sections of E12 treated rats. Submucosal infiltrationof macrophages (ED1+) in damaged areas is shown, and presence ofmacrophages in the lamina propria of unaffected areas is detected. CD3+T cell infiltrate healthy mucosa, with marked IL-10 production at themucosa.

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What is claimed is:
 1. A method of treating an autoimmune inflammatorydisease in a subject in need thereof, the method comprisingadministering to said subject a therapeutically effective amount of anisolated anti-SR-B1 antibody, thereby treating the autoimmuneinflammatory disease in the subject, wherein said autoimmuneinflammatory disease is selected from the group consisting of SystemicLupus Erythematosus (SLE), Amyotrophic Lateral Sclerosis (ALS) andRheumatoid Arthritis (RA).
 2. The method of claim 1, wherein saidisolated anti-SR-B1 antibody reduces secretion of IL-12 or TNF-α or bothin leukocytes.
 3. The method of claim 1, wherein said isolatedanti-SR-B1 antibody increases secretion of IL-10 in leukocytes.
 4. Themethod of claim 1, wherein said isolated anti-SR-B1 antibody reducessecretion of IL-12 or TNF-α or both, and increases secretion of IL-10 inleukocytes.
 5. The method of claim 1, wherein said isolated anti-SR-B1antibody reduces secretion of IL-12 or TNF-α or both in macrophages. 6.The method of claim 1, wherein said isolated anti-SR-B1 antibodyincreases secretion of IL-10 in macrophages.
 7. The method of claim 1,wherein said isolated anti-SR-B1 antibody reduces secretion of IL-12 orTNF-α or both, and increases secretion of IL-10 in macrophages.
 8. Themethod of claim 1, wherein said isolated anti-SR-B1 antibody reducessecretion of IL-12 or TNF-α or both in T cells.
 9. The method of claim1, wherein said isolated anti-SR-B1 antibody increases secretion ofIL-10 in T cells.
 10. The method of claim 1, wherein said isolatedanti-SR-B1 antibody reduces secretion of IL-12 or TNF-α or both, andincreases secretion of IL-10 in T cells.
 11. The method of claim 1,wherein said isolated anti-SR-B1 antibody is a human anti-SR-B1antibody.
 12. The method of claim 1, wherein said autoimmuneinflammatory disease is Systemic Lupus Erythematosus (SLE).
 13. Themethod of claim 1, wherein said autoimmune inflammatory disease isAmyotrophic Lateral Sclerosis (ALS).
 14. The method of claim 1, whereinsaid autoimmune inflammatory disease is Rheumatoid Arthritis (RA).